E. coli O157:H7 C1-INH-binding protein and methods of use

ABSTRACT

Disclosed is a pO157 plasmid-specified polypeptide found in  E. coli  EDL933 and other  E. coli  that binds to and cleaves C1-esterase inhibitor, and antibodies specific for the polypeptide. Also disclosed are methods employing the polypeptide for diagnosing enterohemorrhagic  E. coli  infection, identifying potential inhibitors of its activity, and reducing viscosity of material containing glycosylated polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.10/002,309, filed Oct. 26, 2001, now U.S. Pat. No. 6,872,559, and claimspriority to U.S. Provisional Application No. 60/243,675, filed Oct. 26,2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded byNIH AI051735.

INTRODUCTION

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 strain is ahuman enteric bacterial pathogen that causes diarrheal disease,hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Each year inthe United States, an estimated 20,000 people suffer from diarrhealdisease associated with E. coli 0157:H7 infection, which is typicallycontracted by ingesting contaminated foods, especially undercooked meat.Approximately 6% of infected individuals develop HUS, which can lead torenal failure and death. Young children and the elderly are particularlysusceptible to developing HUS.

In general, bacterial infections are commonly treated by administeringappropriate antibiotics. However, E. coli O157:H7 infection typicallyhas a very rapid progression, and is consequently very difficult totreat. Often by the time the disease is diagnosed, the infectedindividual is severely ill and toxic proteins secreted by the bacteriamay have damaged mucosal cells and entered the blood stream. Antibiotictreatment of patients infected with E. coli O157:H7 is generally notsuccessful and, in fact, is believed to be contraindicated.

E. coli O157:H7 bacteria are very proficient at establishing aninfection; ingestion of as few as 10 live bacteria is sufficient toestablish an infection. The highly infective nature of E. coli O157:H7and the devastating sequelae associated with infection by this bacteria,together with the extensive public attention given to outbreaks ofhemorrhagic colitis, has generated a great deal of interest amongmedical professionals and the general public in developing the means forearly diagnosis and treatment of the disease. The entire genome of theE. coli O157:H7 EDL933W (ATCC 43895) was sequenced with the expectationthat valuable information concerning the organism's pathogenicity wouldbe uncovered, which may facilitate development of methods of preventinginfections, or preventing or treating hemolytic uremic syndrome inindividuals infected with of the organism. The DNA sequence of E. coliO157:H7 was compared with that of E. coli K12, a non-pathogenic straincommonly used in research. The genome of E. coli O157:H7 exceeds that ofE. coli K-12 by more than a million base pairs and has up to 1000 genesnot found on K-12. These additional gene sequences are distributedthroughout more than 250 sites in islands, with each island containingfrom zero to sixty genes (1).

There is a need for improved methods of early detection of E. coliO157:H7 infections and for methods of preventing or treating individualsinfected with E. coli O157:H7.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a purified antibody thatbinds specifically to a polypeptide comprising SEQ ID NO:2.

In another aspect, the present invention includes a method of reducingcolonization of epithelial cells by StcE producing bacteria comprisingcontacting the epithelial cells with an antibody that binds specificallyto SEQ ID NO:2 or an inhibitor of StcE.

In yet another aspect, the invention provides a composition comprising apurified polypeptide comprising at least 25 consecutive amino acidresidues of SEQ ID NO:2 and an adjuvant.

Also provided is a method of eliciting an immune response in an animalcomprising inoculating the animal with a composition comprising apurified polypeptide comprising at least 25 consecutive amino acidresidues of SEQ ID NO:2 and an adjuvant.

The present invention also provides a method of reducingcomplement-mediated disruption of cells comprising contacting the cellswith a purified polypeptide comprising amino acid residues 24–886 of SEQID NO:2 or SEQ ID NO:19 so as to reduce complement-mediated disruptionrelative to that of untreated cells.

The invention further provides a method of reducing the viscosity of amaterial comprising a mucin or a glycosylated polypeptide comprisingcontacting the material with a viscosity reducing effective amount ofStcE.

In another aspect, the invention provides a composition for enhancingdelivery of a target antigen to mucosal cells comprising the targetantigen and StcE.

The composition for enhancing delivery of a target antigen to mucosalcells may be used in a method of eliciting in an animal an immuneresponse to a target antigen comprising contacting the mucosal cells ofthe animal with the composition.

The present invention provides a method of detecting StcE in a sample bydetecting binding of an antibody with selectively for a polypeptidecomprising SEQ ID NO:2 of claim 1 to a polypeptide in the sample.

In another aspect, the invention includes detecting StcE activity bycontacting the sample with C1-INH under suitable conditions to allowcleavage of C1-INH by StcE, if present and detecting C1-INH cleavage.

The invention also provides a method of evaluating a test substance forthe ability to inhibit StcE comprising contacting C1-INH with the testsubstance and StcE and detecting C1-INH cleavage.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the differential effect of E. coli strains containing (FIG.1A and FIG. 1C) or lacking (FIG. 1B and FIG. 1D) the plasmid pO157 onaggregation of T cells.

FIG. 2 shows stained proteins separated by SDS/PAGE.

FIG. 3 shows that synthesis of StcE correlates with the presence ofpO157.

FIG. 4 shows that C1 inhibitor in human serum is cleaved by StcE.

FIG. 5 shows cleavage of C1 inhibitor over time.

FIG. 6 shows differential cleavage of C1-INH by StcE and elastase.

FIG. 7 is an immunoblot showing a StcE-reactive band in fecal filtrates.

FIG. 8A shows that StcE E435D-His is unable to cleave C1-INH; FIG. 8Bshows that StcE E435D-His is unable to bind C1-INH.

FIG. 9 shows amplification of three stcE-specific sequences from all E.coli isolates containing the pO157, and amplification of two of thethree stcE-specific sequences.

FIG. 10 shows an immunoblot of culture supernatants probed with apolyclonal antibody to StcE.

FIG. 11 shows an immunoblot of C1-INH following incubation with culturesupernatants, probed with anti-C1-INH antibody.

FIG. 12 shows the percent lysis of erythrocytes in classicalcomplement-mediated erythrocyte lysis by human serum as a function ofStcE (solid squares) or BSA (open squares) concentration.

FIG. 13A shows the percent lysis of erythrocytes contacted with C1-INHtreated with increasing concentrations of StcE′-His (closed squares) orBSA (open squares) prior to adding human serum. The point indicated by acircle lacked C1-INH. FIG. 13B shows the percent lysis of erythrocytescontacted with C1-INH treated StcE′-His (closed squares) or BSA(opensquares) prior to adding human as a function of C1-INH concentration.

FIG. 14A shows binding of StcE′-His or Alexa-StcE′-His to erythrocytesas detected by flow cytometry. FIG. 14B shows mean fluorescence oferythrocytes detected by flow cytometry as a function of increasingconcentrations of Alexa-StcE′-His.

FIG. 15A shows detection of erythrocyte binding by untreated C1-NH, orC1-INH treated with buffer alone (“mock treated”) or StcE′-His asdetected by flow cytometry. FIG. 15B shows detection of erythrocytebinding by C1-INH treated with StcE′-His, with or without subsequentremoval of StcE′-His, as detected by flow cytometry. FIG. 15C shows meanfluorescence detection of erythrocyte binding by C1-INH treated withStcE′ E435D-His with increasing concentrations of C1-INH. FIG. 15D showsa blot of immunoprecipitated C1-INH untreated or treated with StcE′-Hisor StcE′ E435D-His, separated by SDS-PAGE, transferred tonitrocellulose, and probed with an anti-StcE′ Ab.

FIG. 16A shows the percent lysis of erythrocytes in classicalcomplement-mediated erythrocyte lysis by human serum followingpretreatment with C1-INH, C1-INH and StcE′-His, or C1-INH and StcE′E435D-His. FIG. 16B shows binding of C1-INH was untreated or treatedwith StcE′-His or StcE′ E435D-His before the addition of sheeperythrocytes as detected by flow cytometry.

FIG. 17A shows the relative kallikrein activity in the presence ofincreasing concentrations of C1-INH in the presence or absence ofStcE′-His. FIG. 17B shows an immunoblot of C1s untreated or treated withC1-INH in the absence or presence of StcE′-His or StcE′ E435D-His.

FIG. 18A shows blots of C1-NH untreated or treated with StcE′-His orkallikrein and probed with a polyclonal anti-human C1-INH Ab (leftpanel), mAb 3C7 (middle panel), or mAb 4C3 (right panel). FIG. 18B showselectrophoretically separated ³⁵S-methionine-labeled full length hC1-INHor C-serp(98), a recombinant C1-INH molecule truncated at amino acid 98,each untreated or treated with StcE′-His and immunoprecipitated withpolyclonal anti-human C1-INH IgG-Protein A sepharose.

FIG. 19 shows the percent survival of serum sensitive bacteria in thepresence of serum and of C1-INH, StcE′-His, C1-INH and StcE′-His, or noadditional protein.

FIG. 20 compares the number of pedestals per field formed for wild type,StcE knockout, and complemented E. coli O157:H7 strains on HEp-2 cellsin the presence or absence of exogenous StcE.

FIG. 21 compares the viscosity of-saliva before (zero hour) and afterincubation in buffer (squares) or in StcE (triangles) relative to water(dashed line) as measured by elution time as a function of incubationtime.

FIG. 22 shows human salival proteins separated by SDS-PAGE and stainedwith Coomassie.

FIG. 23 is a graph showing the concentration of detectable C1-INH in awell as a function of the amount of C1-INH added to the well for samplestreated with StcE in the presence (squares) or absence (triangles) ofEDTA.

DETAILED DESCRIPTION OF THE INVENTION

Each of the publications or applications cited or listed herein isincorporated by reference in its entirety.

Strains of the serotype O157:H7 EDL933W contain a 92 kb plasmiddesignated pO157. As described in the Examples below, bacterial strainscontaining the plasmid cause the aggregation of two cultured human CD4⁺T cell lines, Jurkat and MOLT-4, but do not cause aggregation of a Bcell lymphoma line (Raji), or of macrophage-like cell lines (U937 andHL-60). Aggregation of the CD4⁺ T cells occurs in the presence of serum,but not in its absence. Strains lacking the plasmid do not causeaggregation of CD4⁺ T cells.

We employed transposon mutagenesis to identify a gene on pO157 ofpreviously unknown function whose product is associated with theobserved aggregation effect. The coding sequence and the deduced aminoacid sequence of the protein it encodes are shown in SEQ ID NO:1 and SEQID NO:2, respectively. The protein, designated “StcE”, contains aputative cleavable N-terminal signal sequence. In cultures of bacteriaexpressing StcE, at least a portion of expressed StcE protein appears tobe secreted into the culture medium, although StcE may also beassociated with the cell surface.

A genetic construct expressing a His-tagged StcE protein was made byligating the StcE coding sequence in frame to a sequence specifying apoly-His tail to permit expression and recovery of relatively largeamounts of highly purified StcE protein, as described below.

In order to further evaluate the StcE protein for possible cytotoxiceffects, variety of cell types were treated with StcE protein asdescribed in the examples. Cells treated with StcE showed a high degreeof aggregation in the presence of serum, but not in the absence ofserum.

Because StcE-mediated aggregation occurred only in cells also treatedwith serum, the ability of StcE to bind to a specific serum protein wasevaluated by Far Western blotting using the StcE protein as the probe.An acidic serum protein of about 105 kDa by SDS PAGE was identified asbinding to StcE. The target protein was recovered, subjected to limiteddigestion by an endopeptidase, and the peptide products analyzed by massspectrometry. The protein to which StcE binds was identified asC1-inhibitor (C1-INH), which serves as a critical inhibitor in theproteolytic cascade involved in complement activation.

The plasma protein C1-INH is a serpin (serne protease inhibitor) thatcontrols the activation of C1, the first component of the complementcascade. The C1 component is made up of three subcomponents: C1q, C1r,and C1s. In the classical pathway of complement activation, C1 binds toan antigen-antibody complex or certain pathogens (e.g., HIV-1), whichcauses the proteolytic autoactivation of C1r, which in turn causes theproteolytic activation of C1s. C1-INH inhibits activation of theclassical pathway by binding to C1 and inactivating C1r and C1s. Inaddition to its role in controlling activation of the classicalcomplement pathway, C1-INH inhibits other serine proteases involved inthe intrinsic coagulation pathway and kinin-forming system (reviewed in(3)).

Treatment of serum or purified C1-INH with purified StcE results in theapparent disappearance of C1-INH, presumably as a result of specificproteolytic cleavage of C1-INH by StcE. The predicted StcE amino acidsequence comprises the sequence HEVGHNYGLGH (SEQ ID NO:3) (residues434–444 of SEQ ID NO:2), which corresponds to the histidine rich activesite of metalloproteases (5). Further evidence that StcE may be ametalloprotease is provided by the observation that proteolysis ofC1-INH by StcE is reduced in the presence of EDTA or BPS, which chelatedivalent metal ions (e.g., Zn²⁺) required for metalloprotease activity.

In U.S. application Ser. No. 10/002,309, filed Oct. 26, 2001, of whichthe present application is a continuation-in-part, we proposed that theputative protease StcE by enterohemorrhagic strains of E. coli EHEC maylead to proteolysis of C1-INH and reduction of C1-INH activity. Loss ofC1-INH activity may result in unregulated pro-inflammatory orcoagulation response that may be responsible for tissue damage in theintestine and kidney of persons infected with EHEC. It is also possiblethat the StcE serum-dependent cellular aggregation phenotype plays arole in the pathogenesis of HUS because one of the hallmarks of HUS isthrombocytopenia with an accumulation of a large number of platelets inrenal microthrombi. The kidneys of those diagnosed with HUS also containlarge amounts of deposited fibrin.

Deficiencies in C1-INH can lead to a variety of diseases. For example, ahereditary deficiency in C1-INH (hereditary angiodema) is characterizedby transient, recurrent attacks of intestinal cramps, vomiting anddiarrhea. Hereditary defects in production of a different inhibitor ofthe complement cascade, Factor H, are associated with a form ofhemolytic uremic syndrome (HUS) similar to that described forEHEC-mediated HUS.

The proteolytic activity of StcE may be a common mode of pathogenesisamong some diarrheagenic strains of E. coli. Colony blot analysis andamplification of E. coli DNA using oligomers specific to the pO157version of stcE indicate that the stcE gene is common to all testedstrains of E. coli associated with bloody colitis and HUS, but the stcEgene is not present in enteroinvasive, enterotoxigenic or uropathogenicstrains of E. coli. However, some closely related strains ofenteropathogenic E. coli contain stcE, which suggests that StcE may bemore widely distributed among diarrheagenic E. coli than appreciatedinitially. Additionally, a search of the GenBank database has identifiedat least one distant homolog to StcE: a Vibrio cholerae protein(designated TagA) of unknown function. We envision methods to screen forsimilar virulence factors produced by other microbes.

We discovered that in the presence of cells, StcE surprisinglypotentiates the ability of C1-INH to reduce complement-mediateddisruption of cells. Rather than reducing or destroying C1-INH serpinactivity, the interaction between cell-associated StcE and C1-INHappears to enhance the ability of C1-INH to inhibit the classicalcomplement cascade, thereby protecting cells from the lytic effects ofcomplement activity. As detailed in the Examples, the addition ofStcE-treated C1-INH to opsonized sheep erythrocytes and human serumsignificantly decreases erythrocyte lysis below that of equivalentamounts of native C1-INH alone. A decrease in complement activity whenhuman serum was treated with StcE prior to the addition of sheeperythrocytes was also observed, which is likely due to the activity ofStcE on the endogenous C1-INH found in serum. Furthermore, analysis ofcomplement component deposition on erythrocyte surfaces indicates thatStcE-treated C1-INH continues to act on its natural targets C1r and/orC1s.

StcE is unable to potentiate C1-INH activity in the absence of cells.Rather than directly modifying C1-INH to increase its ability to inhibitthe complement cascade, StcE potentiation of C1-INH in the presence ofcells may be due to tethering C1-INH to the cell surface, effectivelyincreasing the local concentration of C1-INH at the sites of potentiallytic complex formation.

Cleavage of C1-INH by StcE does not appear to be a factor in StcEpotentiation of C1-INH protection against complement activity, as isevidenced by the ability of StcE E435D-His, a mutant protein defectivein proteolytic activity against C1-INH, to protect erythrocytes to adegree comparable to that observed with wild-type StcE in vitro. Theinteraction between StcE and erythrocytes is specific, saturating thecells at approximately 1.8×10⁶ molecules of StcE per cell. In turn, thisallows a high affinity interaction between C1-INH and erythrocytes,reaching 2.25×10⁶ molecules of C1-INH per cell in the presence of one μgof StcE. This amount of C1-INH (0.1 IU) is well within the physiologicalconcentration of C1-INH found in serum, suggesting that this interactionmay be biologically relevant in vivo.

Without being limited as to theory, we propose a model of the mechanismby which StcE potentiates C1-INH-mediated inhibition of classicalcomplement in which StcE first interacts with the cell surface, followedby binding of StcE to the N-terminal domain of C1-INH to sequester theserpin to the cell surface. Alternatively, StcE may interact with C1-INLbefore binding to the cell surface. Cell-bound C1-INH binds to C1rand/or C1s of the C1 complex via the RCL, inactivating the serineprotease. StcE then cleaves within the N-terminus of C1-INH, releasingthe serpin/serine protease complex and a smaller, amino-terminalcleavage fragment of C1-INH from the cell surface. StcE binds to anotherC1-INH molecule, and the cycle described above is repeated.

The concept of C1-INH turnover at the cell surface may explain theobservation that more C1-INH bound to erythrocytes in the presence ofthe proteolytically inactive StcE mutant than was bound in the presenceof StcE′-His. Thus, the relatively slow cleavage rate of C1-INH by StcEmay actually be beneficial to the potentiation of the serpin, as a morerapid rate of catalysis might not allow enough time for the inactivationof the C1 complex to occur. Additionally, the interaction of StcE withthe N-terminal region of C1-INH while bound to the cell surface likelypermits continued access of the serpin domain to its targets withoutcompromising its activity. The binding of StcE to host cells might alsoallow the protease to be carried to sites distal to E. coli O157:H7colonization in a manner similar to the Shiga toxin, thereby affectingC1-INH-regulated processes outside the local environment of bacterialinfection. Finally, the observation that cell-bound C1-INH might affectleukocyte adhesion suggests StcE could influence the migration ofinflammatory mediators to the sites of EHEC colonization.

Sequestration of complement inhibitors to bacterial surfaces can reducecomplement activity and promote serum resistance. As demonstrated in theExamples, StcE-treated C1-INH acts in a similar by providing increasedserum resistance to E. coli over native C1-INH. By preventing complementactivity at such an early stage in the pathway via the recruitment ofC1-INH to cells, StcE may also reduce the opsonization of E. coliO157:H7 by C3b and the production of the chemoattractant anaphylatoxinsC3a and C5a. Because StcE is a secreted protein, the protection againstcomplement activation may extend to the colonic epithelial cellscolonized by E. coli O157:H7.

Although the Examples indicate that StcE has no effect onC1-INH-mediated inhibition of kallikrein in the absence of cells, it isreasonably expected that StcE-treated C1-INH may downregulate contactactivation pathway initiated upon the interaction of Factor XII andprekallikrein with negatively charged surfaces.

In addition to its ability to sequester C1-INH to cell surfaces andcleave in the N-terminal region of C1-INH, we have discovered that StcEis capable of cleaving heavily glycosylated polypeptides and mucins,such as those found in saliva or sputum, including, but not limited to,MUC7 and gp-340/DMBT1.

The ability of StcE to cleave glycosylated polypeptides may facilitatecolonization of the gut by E. coli O157:H7. The Examples below show thata StcE knockout mutant has impaired ability to form pedestals, butpedestal formation is restored by supplementation with exogenous StcE.

Elucidation of StcE functions suggests that StcE plays a role in theestablishment and progression of EHEC infection and associated diseaseprocesses. StcE is therefore a promising potential target forchemotherapeutic or immune-based prevention or treatment of EHECdiseases. Active or passive immune prophylaxis using StcE as an antigenor anti-StcE antibodies may prevent the serious sequalae associated withinfections by enterohemorrhagic E. coli.

The development of an assay for StcE activity as described herein belowwill facilitate screening of potential therapeutics for the ability toinhibit cleavage of the C1-INH.

Preferably, purified polypeptides will be used in the methods of thepresent invention (i.e., to assay potential StcE inhibitors or to elicitan immune response in an animal). As used herein, a purified peptide isat least 95% pure, in that it contains no greater than 5% non StcEpeptide sequences. More preferably, purified StcE polypeptides are atleast 97% pure, or even as much as at least 99% or more pure. Purifiedpolypeptides may be obtained by standard biochemical purification means,or by engineering recombinant proteins to include affinity tags thatfacilitate purification from complex biological mixtures.

In the examples below, polyclonal antibodies were raised against aHis-tagged StcE protein that was first purified by immobilized metalaffinity chromatography (IMAC) using nickel-agarose beads, followed byseparation by SDS-PAGE, and excision of the band of the appropriate sizefrom the gel. The antibodies were found to bind specifically to StcEpolypeptide. One of skill in the art will appreciate that using standardmethods, monoclonal antibodies useful in the practice of the presentinvention could be raised against a polypeptide comprising the aminoacid sequence of amino acid residues 24–886 of SEQ ID NO:2 or againstshorter consecutive peptide sequences thereof.

It is expected that antibodies directed against StcE will bind to andreduce StcE activity. For example, it is expected that includinganti-StcE antibodies in assays of the ability of StcE-producing strainsof E. coli to form pedestals would result in reduced pedestal formation.Similarly, anti-StcE antibodies could be expected to reverseStcE-potentiated C1-INF protection of cells from disruption bycomplement activation, to interfere with cleavage of StcE by C1-INH in acell-free assay, and to interfere with the ability of StcE to cleaveother heavily glycosylated polypeptides.

It is envisioned that an antibody preparation comprising at least oneantibody that binds specification to a polypeptide comprising amino acidresidues 24–886 of SEQ ID NO:2 could be used to passively immunize ananimal at risk of infection by a bacterium expressing a StcE protein.This would be particularly useful for treating cows or humans believedto have been exposed to EHEC.

In view of the multiple functions that StcE appears to play inprotecting the bacteria against or overcoming host defense mechanisms,purified polypeptides comprising a sequence of at least 17, 25, or 40consecutive amino acids of SEQ ID NO:2 may be particularly useful as animmunogen for eliciting an immune response in cattle or humans.Suitably, the full length StcE or StcE E435D could be used as animmunogen.

The Examples below show that StcE is able to cleave mucins or otherglycosylated polypeptides to reduce the viscosity of or solubilize thematerials. It is specifically envisioned that the StcE could be used tocleave glycosylated polypeptides, thereby reducing the viscosity of thematerial comprising the polypeptides. The ability of StcE to cleaveglycosylated polypeptides and reduce viscosity of sputum or pulmonarysecretions would be of potential benefit to people with cystic fibrosis.

It is also envisioned that the ability of StcE to cleave glycosylatedpolypeptides and reduce the viscosity of saliva and other mucousmaterials would make it useful in a mucosal vaccine in conjunction witha target antigen of interest (i.e., an antigen against which one wishesto illicit an immune response in an animal) because it may enhanceaccess of the target antigen to target cells.

As one of skill in the art would appreciate, a StcE protein comprisingan amino acid sequence having minor substitutions, deletions, oradditions from that of the SEQ ID NO:2 would be suitable in the practiceof the present invention. Conservative amino acid substitutions areunlikely to perturb the protein's secondary structure and interfere withits activity. SEQ ID NO:2 includes the N-terminal signal sequence, whichalthough expressed, is unlikely to be found on an isolated polypeptide.The expressed StcE protein likely undergoes post translationalmodification that results in cleavage of the N-terminal signal peptide.The N-terminus of secreted StcE was purified from supernatants of an E.coli K-12 strian carrying p0157 was sequenced. As predicted, theN-terminal residues of secreted StcE (designated StcE′) correspond toresidues 24–27 of SEQ ID NO:2.

It is specifically envisioned that isolated polypeptides having lessthan the full length sequence of amino acid residues 24–886 of SEQ IDNOL:2 will be useful in the practice of the present invention. StcEpolypeptides that are truncated at the N-terminal or C-terminal regionsor having minor sequence variations may retain the ability to bind toand/or cleave C1-INH, to promote pedestal formation, to potentiateC1-INH mediated protection of cells, or cleave other glycosylatedpolypeptides other than or in addition to C1-INH. Whether a proteinretains binding or proteolytic activity of can be evaluated using themethods set forth herein in the Examples, or by any suitable method.

It is expected that purified StcE polypeptides that are truncated at theN-terminal or C-terminal regions or a polypeptide comprising an aminoacid sequence comprising at least 17 consecutive amino acid residues ofSEQ ID NO:2 may be used as an antigen against which antibodies specificfor StcE may be raised. Preferably, the polypeptide comprises at least25 consecutive amino acid residues of SEQ ID NO:2. More preferablystill, the polypeptide comprises at least 40 consecutive amino acidresidues of SEQ ID NO:2. One of ordinary skill in the art could easilyobtain any of the various polypeptides comprising a portion of SEQ IDNO:2 by subcloning a sequence encoding the polypeptide into anexpression vector, introducing the expression vector into a suitablehost cell, culturing the cell, and isolating the expressed polypeptideusing standard molecular biological techniques.

Using the teachings of the specification, one of skill in the art couldreadily obtain a polypeptides having at least 95% amino acid identity toamino acid residues 24–886 of SEQ ID NO:2. Suitably, the polypeptidecomprises a sequence having at least 97% or at least 99% amino acididentity to amino acid residues 24–886 of SEQ ID NO:2.

The following non-limiting examples are intended to be purelyillustrative.

EXAMPLES

Identification and Characterization of StcE

A list of bacterial strains and plasmids is found in Table 1. Strainswere constructed and plasmids were maintained in either E. coli K-12 DH1or C600 unless otherwise noted. Recombinant DNA manipulations wereperformed by standard methods.

Enterohemorrhagic Escherichia coli strains EDL933 and EDL933cu (lackingplasmid pO157) and WAM2371 (enteropathogenic E. coli strain E2348/69)were provided by Dr. Alison O'Brien of the Uniformed ServicesUniversity. WAM2035 (C600/pO1 57) was provided by Dr. Hank Lockman ofthe Uniformed Services University. WAM2516 (Citrobacter rodentium strainDBS100) was provided by Dr. David Schauer of the Massachusetts Instituteof Technology. The Diarrheagenic E. coli (DEC) collection was a giftfrom Dr. Tom Whittam of the University of Pennsylvania. WAM2547 wascreated by transforming pLOF/Km (a gift from Dr. Victor De Lorenzo ofthe GBF-National Research Centre for Biotechnology, Germany) into thedonor strain S17(λpir).

TABLE 1 Bacterial strains and plasmids used in this study. StrainRelevant phenotype or plasmid genotype Source C600 E. coli K-12 thislaboratory DH1 laboratory strain of E. coli this laboratory S17(λpir) E.coli donor strain for conjugation this laboratory BL21(DE3) E. colistrain for protein overexpression Novagen EDL933 wild-type EHEC strainA. O'Brien EDL933cu EHEC strain EDL933 cured of pO157 A. O'Brien WAM2371EPEC strain E2348/69 A. O'Brien WAM2516 C. rodentium strain DBS100 D.Schauer DEC strains Diarrheagenic E. coli collection T. Whittam WAM2035C600/pO157::Tn801 (amp^(r)) H. Lockman WAM2515 C600/pO157::Tn801(amp^(r) nal^(r)) this study WAM2297 DH1/pBluescript II SK+ (amp^(r))this laboratory WAM2547 S17(λpir)/pLOF/Km (amp^(r) kan^(r)) this studyWAM2553 C600/pWL104 (amp^(r) kan^(r)) this study WAM2562 DH1/pWL105(amp^(r)) this study WAM2572 BL21(DE3)/pWL107 (kan^(r)) this studyWAM2726 BL21(DE3)/pTEG1 (kan^(r)) this study WAM2815 EDL933 with stcEreplaced by cat this study WAM2997 WAM2815 strain with stcE at theTn7att site this study pLOF/Km pGP704 carrying miniTn10kan V. De LorenzopO157 92 kb plasmid of EDL933; Tn801 at base 5413 H. Lockman pBluescriptII SK+ cloning vector Stratagene pET24d(+) 6xHis overexpression vectorNovagen pWL104 pO157::miniTn10kan inserted at base 23772 this studypWL105 pBluescript II SK+/bases 1–2798 of L7031 this study pWL107pET24d(+)/bases 138–2795 of L7031 this study pTEG1 pWL107 with aminoacid change E435D this study

WAM2515 is a spontaneous nalidixic acid-resistant mutant of WAM2035.WAM2553 was created as described below, containing a mini-Tn10kaninsertion at base 23772 of pO157 (accession #AF074613). This plasmid isdesignated pWL104. WAM2297 is pBluescript II SK+ in DH 1. pWL105 wasconstructed by amplifying bases 1 to 2798 of the promoter and geneL7031/stcE from pO157 by polymerase chain reaction (PCR) using primerpairs 5′-CCCTCGAGTTTACGAAACAGGTGTAAAT-3′ (SEQ ID NO:4) and5′-CCTCTAGATTATTTATATACAACCCTCATT-3′ (SEQ ID NO: 5); and cloning theproduct into the XbaI-XhoI sites of pBluescript II SK+ (Stratagene);WAM2562 is DH1 containing pWL105. pWL107 was constructed by PCRamplification of bases 138 to 2798 of the promoter and gene L7031/stcEfrom pO157 by PCR using primer pairs5′-CCGAGCTCCGATGAAATTAAAGTAT-CTGTC-3′ (SEQ ID NO:6) and5′-CCTCGAGTTTATATACAACCCTCATTG-3′ (SEQ ID NO:7); and cloning the PCRproduct into the SacI-XhoI sites of pET-24d(+) (Novagen); WAM2572 isBL21(DE3) (Novagen) transformed with pWL 107. The creation of WAM2726 isdescribed below. All chemicals were purchased from Sigma (St. Louis,Mo.) unless stated otherwise.

Cell Lines

All cell lines were maintained in RPMI 1640 medium (Gibco) supplementedwith 10% fetal bovine serum (HyClone) and 10 μg/ml gentamicin at 37° C.with 5% CO₂. The human T cell line Jurkat clone E6-1, the humanpromyelocytic leukemia line HL-60, and the human B cell lymphoma lineRaji were obtained from ATCC, the human promyelocytic leukemia line U937was a gift from Dr. Jon Woods of the University of Wisconsin-Madison,and the human T cell lymphoma line MOLT-4 was a gift from Dr. DavidPauza of the University of Wisconsin-Madison.

Aggregation Assays

Bacterial strains were grown overnight in Lennox L broth (withantibiotic selection when appropriate) at 37° C. with agitation.Cultures were washed once with phosphate buffered saline (PBS) andresuspended in 1/10 the original culture volume in PBS. Cultures werelysed in a French Press at 20,000 lbs/in². The resulting lysates werespun at 1000×g to remove debris and protein concentrations weredetermined by the Bradford protein assay (Bio-Rad). Tissue culture cellswere suspended at 10⁶ cells/ml in RPMI 1640 and 50 μg/ml gentamicin with10% FBS or human serum. Fifty μg/ml of lysates or 50–200 ng/ml purifiedStcE-His (see below) were added to cells and incubated for two hours at37° C. in 5% CO₂. Cells were agitated for one minute to disruptspontaneous aggregates before visualization. Similar assays wereperformed in the absence of serum or with ammonium sulfate-precipitatedfractions of human serum (see below); cells were washed once in RPMI1640 and resuspended at 1×10⁶ cells/ml in RPMI 1640 with 50 μg/mlgentamicin (and human serum fractions, if indicated) before the additionof lysates or StcE-His. When indicated, ethylenediaminetetraacetic acid(EDTA) or bathophenanthroline-disulfonic acid (BPS) were added to theassays at a final concentration of 5 mM.

Identification of StcE

WAM2515 was mated with WAM2547 as described (7). Transconjugants wereplated onto LB plates containing 100 μg/ml ampicillin, 50 μg/mlkanamycin, and 50 μg/ml nalidixic acid. Transconjugants were resuspendedin 1× TES, washed once with 1×TES, and pO157/pO157::mini-Tn10kan wereisolated by midi-prep (Qiagen). pO157/pO157::mini-Tn10kan weretransformed into C600 and plated onto LB plates containing 100 μg/mlampicillin and 50 μg/ml kanamycin. Transformants were grown overnight inLennox L broth containing 100 μg/ml ampicillin and 50 μg/ml kanamycin at37° C. with agitation and lysates were screened for the ability toaggregate Jurkat cells as described above. pO157::mini-Tn10kan wasisolated from clones lacking the ability to aggregate Jurkat cells andthe location of the transposable element was identified by sequenceanalysis. One clone unable to aggregate Jurkat cells was designatedWAM2553.

Purification of Recombinant StcE-His

StcE-His was purified according to the manufacturer's instructions(Novagen). Briefly, WAM2572 was induced to produce StcE-His by theaddition of IPTG to 1 mM at an O.D. of 0.5 followed by vigorous aerationat 37° C. for approximately three hours. The cells were lysed in aFrench Press at 20,000 lbs/in² and the resulting lysate was centrifugedat 20,000×g for 15 minutes. The insoluble pellet was resuspended in abuffer containing 5 mM imidazole and 6 M urea and the inclusion bodieswere solubilized for one hour on ice. This fraction was incubated withnickel-agarose beads (Qiagen) overnight at 4° C., and the beads werewashed three times with a buffer containing 60 mM imidazole and 6 Murea. Purified StcE-His was eluted from the beads with a buffercontaining 300 mM imidazole and 6 M urea. Eluted StcE-His was dialyzedagainst three changes of PBS/20% glycerol at 4° C. to remove theimidazole and urea. Protein concentration was determined by SDS-PAGEusing purified β-galactosidase as a standard. At our request, polyclonalantibodies to purified StcE-His were prepared in rabbits by CocalicoBiologicals, Inc. Briefly, purified StcE-His was electrophoresed on an8% polyacrylamide gel and stained with Coomassie Brilliant Blue.StcE-His was excised from the gel and injected into rabbits. Rabbitswere boosted with StcE-His once a month for six months prior toexsanguinations.

Two-Dimensional Gel Electrophoresis

Human serum was fractionated by ammonium sulfate precipitation, dialyzedagainst three changes of RPMI 1640 (Gibco) overnight at 4° C., andprotein concentration was determined by Bradford assay (Bio-Rad). Whenindicated, protein A-sepharose was used to remove fractions of IgG.Two-dimensional electrophoresis was performed according to the method ofO'Farrell (8) by Kendrick Labs, Inc. (Madison, Wis.) as follows:isoelectric focusing was carried out on 25 μg of 30–60% ammoniumsulfate-fractionated human serum removed of IgG in glass tubes of innerdiameter 2.0 mm using 2.0% pH 3.5–10 ampholines (Amersham PharmaciaBiotech) for 9600 volt-hrs. Fifty ng of an IEF internal standard,tropomyosin, was added to each sample. This protein migrates as adoublet with lower polypeptide spot of MW 33,000 and pI 5.2; an arrow onthe stained gel marks its position. The enclosed tube gel pH gradientplot for this set of ampholines was determined with a surface pHelectrode.

After equilibration for 10 min in buffer “0” (10% glycerol, 50 mMdithiothreitol, 2.3% SDS and 0.0625 M Tris, pH 6.8) each tube gel wassealed to the top of a stacking gel that is loaded on the top of a 8%acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis wascarried out for about 4 hrs at 12.5 mA/gel. The following proteins(Sigma) were added as molecular weight standards to a well in theagarose which sealed the tube gel to the slab gel: myosin (220 kDa),phosphorylase A (94 kDa), catalase (60 kDa), actin (43 kDa), carbonicanhydrase (29 kDa), and lysozyme (14 kDa). These standards appear asbands on the basic edge of the special silver-stained (O'Connell andStults 1997) 8% acrylamide slab gel. The gel was dried between sheets ofcellophane with the acidic edge to the left.

A similar gel was run as described above with the following differences:250 μg of 30–60% ammonium sulfate-fractionated human serum was loadedonto the IEF gel; the second dimension was run on a 10% acrylamide slabgel and stained with Coomassie Brilliant Blue.

Far Western Blot Analysis

One hundred μg of 30–60% ammonium sulfate-fractionated human serum wasrun on a two-dimensional gel as described above but without staining.After slab gel electrophoresis the gel for blotting was transferred totransfer buffer (12.5 mM Tris, pH 8.8, 86 mM glycine, 10% methanol) andtransblotted to PVDF membrane overnight at 200 mA and approximately 50volts/gel. The PVDF membrane was blocked with 2% milk (Difco) in bufferAD (20 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 0.01% Tween-20) at 4°C. Two μg/ml purified StcE-His was added to the PVDF membrane andallowed to incubate two hours at 4° C. The membrane was washed withbuffer AD and blocked with 2% milk in buffer AD. The membrane wasreacted with polyclonal anti-His antibody conjugated with horse-radishperoxidase (Santa Cruz), washed with buffer AD, and developed with theLumiGlo chemiluminescence detection system (Kirkegaard & PerryLaboratories). The PVDF membrane was then stripped (62 mM Tris, pH 6.8,2% SDS, 10 mM β-mercaptoethanol (β-ME), 30 min, 50° C.), washed withbuffer AD, reacted as above with only the His-HRP antibody, anddeveloped.

Mass Spectrometry

Of the three spots in human serum that reacted with purified StcE-His asidentified by Far Western blotting, only the leftmost spot (the mostacidic) of approximately 100 kDa was accessible for analysis by massspectrometry. This spot was cut from the Coomassie BrilliantBlue-stained 10% slab gel and sent to the Protein Chemistry CoreFacility at the Howard Hughes Medical Institute/Columbia University foranalysis. The spot was digested with endoproteinase Lys-C and analyzedby MALDI-MS. The peptide pattern was compared against known humanproteins in the SWISS-PROT database and was identified as plasmaprotease C1 inhibitor.

Electrophoresis and Immunoblot Analyses

Fifty μg whole and ammonium sulfate-precipitated human serum fractionswere incubated with 500 ng purified StcE-His in 500 μl buffer AD for twohours at room temperature and precipitated with 10% trichloroacetic acid(TCA) on ice for one hour. Precipitates were collected bycentrifugation, resuspended in 1× sample buffer (2% SDS, 10% glycerol,5% β-ME, 1 mM bromophenol blue, 62 mM Tris, pH 6.8), and heated to95–100° C. for 5 min prior to electrophoresis on 8% polyacrylamide gels.Separated proteins were transferred to Hybond ECL nitrocellulose(Amersham Pharmacia Biotech) as described (9) for immunoblot analysis.Blots were blocked with 5% milk in TBST (154 mM NaCl, 20 mM Tris, pH7.6, 0.1% Tween-20), probed with a polyclonal anti-C1 inhibitor antibody(Serotec) and then with HRP-conjugated anti-rabbit secondary antibody(Bio-Rad) before developing as described above.

Sixteen μg purified C1 inhibitor (Cortex Biochem) were incubated with4.8 μg purified StcE-His in 480 μl buffer AD at room temperature; 30 μlof the reaction were removed at various time points, suspended in 1×sample buffer, and heated to 95–100° C. for 5 min prior toelectrophoresis on 8% polyacrylamide gels. Separated proteins weretransferred to nitrocellulose and reacted with anti-C1 inhibitorantibody as described above.

EDL933, EDL933cu, WAM2035, and WAM2553 were grown in Lennox L broth at37° C. overnight, centrifuged, and the culture supernatant was removed.The supernatant was precipitated with ammonium sulfate and the 0–60%fraction was resuspended at 1/100 the original culture volume anddialyzed against three changes of PBS overnight at 4° C. Twenty μl ofthe dialyzed supernatants and 30 μg of EDL933, EDL933cu, WAM2035, andWAM2553 lysates were suspended in 1× sample buffer and heated to 95–100°C. for 5 min prior to electrophoresis on 8% polyacrylamide gels.Separated proteins were transferred to Hybond ECL nitrocellulose andreacted with polyclonal anti-StcE-His antibody, followed byanti-rabbit-HRP secondary antibody.

Casein Proteolysis Assay

Various concentrations of StcE-His were incubated with BODIPYFL-conjugated casein for various times using the EnzChek Protease AssayKit (Molecular Probes, Inc.) and the increase in fluorescence wasmeasured with a fluorimeter as per the manufacturer's instructions.

Lysates of E. coli Strains Carrying pO157 Induce the Aggregation ofTransformed Human T Cell Lines in a Serum-Dependent Manner.

To determine the consequence of pO157-containing E. coli products onJurkat cells, a human T cell lymphoma line, 50 μg/ml of lysates ofstrains EDL933, EDL933cu, WAM2035, WAM2371, WAM2516, and C600 wereapplied to 1×10⁶ Jurkat cells/ml in RPMI 1640 with 10% FBS and 50 μg/mlgentamicin for two hours at 37° C. in 5% CO₂. After agitation for oneminute to disrupt spontaneous aggregates, Jurkats were observed for theinduction of aggregation. Lysates of E. coli strains carrying pO157induced the aggregation of Jurkat cells while lysates of strains lackingpO157 did not (FIG. 1). Lysates of other pathogenic bacteria such asenteropathogenic E. coli strain E2348/69 (WAM2371) and C. rodentium(WAM2516) capable of inducing the attaching and effacing (A/E) phenotypeon intestinal epithelial cells and carrying large virulence plasmidsdifferent from pO157 were unable to induce the aggregation of Jurkatcells. To determine whether this effect was specific for Jurkat cells orcould induce the aggregation of a broader host cell range, 1×10⁶cells/ml in RPMI 1640 with 10% FBS and 50 μg/ml gentamicin of anotherhuman T cell lymphoma line, MOLT-4, two human promyelocytic leukemiacell lines, HL-60 and U937, and a human B cell lymphoma line, Raji, weretreated with 50 μg/ml of EDL933 and WAM2035 lysates for two hours at 37°C. in 5% CO₂. pO157-containing lysates aggregated MOLT-4 cells but notHL-60, U937, or Raji cells (data not shown), indicating T cellspecificity for the phenotype.

To determine the serum requirement for the induction of aggregation, 50μg/ml of lysates of EDL933 and WAM2035 were applied to 1×10⁶ Jurkatcells/ml with 10% human serum and 50 μg/ml gentamicin for two hours at37° C. in 5% CO₂. As seen with FBS, pO157-containing lysates were ableto induce the aggregation of Jurkat cells in the presence of humanserum. However, EDL933 and WAM2035 lysates were unable to induce theaggregation of Jurkat cells under the same conditions in the absence ofserum. To further characterize the component(s) of human serumresponsible for mediating Jurkat cell aggregation in the presence ofStcE, we fractionated human serum by ammonium sulfate precipitationfollowed by dialysis in RPMI 1640. We found that 0–30% and 30–60%, butnot 60–100%, ammonium sulfate-precipitated human serum was able tomediate aggregation of Jurkat cells in the presence of StcE. Thisindicates a factor or factors in serum is required for the aggregationof Jurkat cells when treated with lysates of pO157-containing bacteria.

Identification and Cloning of StcE

To localize the gene(s) on pO157 responsible for the induction ofaggregation of human T cell lines, we subjected pO157 to mutagenesisusing a minitransposon. Lysates of recombinant strains of E. colicontaining pO157 mutagenized with mini-Tn10kan were tested for theability to aggregate Jurkat cells in RPMI 1640 with 10% FBS and 50 μg/mlgentamicin. pO157::mini-Tn10kan was isolated from clones whose lysateswere unable to induce the aggregation of Jurkat cells. The location ofthe transposon insertion in WAM2553 was determined by sequence analysisand mapped to position 23772 of pO157. The open reading frame in whichthe transposon inserted was designated L7031 (10) and is locatedimmediately 5′ to the general secretory apparatus on pO157. L7031/stcEwas amplified and cloned into the XbaI-XhoI sites of pBluescript II SK+.Lysates of WAM2562 induced aggregation of Jurkat cells in the presenceof serum, whereas lysates of WAM2297 (DH1 carrying pBluescript II SK+)did not, which confirms that the stcE gene is responsible for thephenotype.

Based on sequence analysis, we concluded that the translational startsite for StcE was more likely to begin at base 138 than at base 102(10). We therefore amplified the coding sequence for stcE from bases 138to 2798 by PCR and cloned the gene in frame with a 6×His-tag at the 3′end of the fusion in pET24d(+). We were able to overexpress and purify arecombinant his-tagged form of StcE (StcE-His) (FIG. 2); this purifiedfusion protein was able to aggregate Jurkat cells in the presence ofserum at a variety of concentrations (data not shown).

Localization and Characterization of StcE

Using antiserum to StcE-His, we performed immunoblot analysis to examinethe expression and secretion of StcE by E. coli. StcE is expressed by E.coli strains carrying pO157 at 37° C. in Lennox L broth but not instrains lacking pO157 or harboring a transposon insertion in stcE (FIG.3). Additionally, StcE is released into the culture supernatant bystrains carrying pO157 under the same growth conditions (FIG. 3). AsStcE contains a putative cleavable N-terminal signal sequence, it ispossible that StcE is actively released from the bacterium by thegeneral secretory apparatus encoded on pO157.

StcE-mediated Jurkat cell aggregation is inhibited by the addition ofion chelators such as EDTA, a broad chelator of divalent cations, andBPS, a chelator specific for zinc and iron ions (data not shown). Thissuggests that StcE has a requirement for one or more divalent cations,most likely zinc. This is supported by the presence of an exact match tothe histidine-rich consensus active site for metalloproteases, whichcoordinate zinc ions for activity (see discussion).

StcE-His interacts with a human serum protein(s) of approximately 105kDa. To identify the factor(s) in human serum responsible for mediatingJurkat cell aggregation in the presence of StcE, the 30–60% ammoniumsulfate-precipitated fraction of human serum was separated on atwo-dimensional gel and transferred to a PVDF membrane. Using purifiedStcE-His as a probe, we performed Far Western blot analysis on the PVDFmembrane, detecting any interactions between StcE-His and human serumproteins with an HRP-conjugated anti-His antibody. We found thatStcE-His interacts with three spots of approximately 105 kDa rangingfrom very acidic to very basic in isoelectric point (data not shown).Probing the same membrane with only the HRP-conjugated anti-His antibodyrevealed that the three spots of approximately 105 kDa were specific forStcE-His (data not shown).

To identify these proteins, the 30–60% ammonium sulfate-precipitatedfraction of human serum was removed of IgG and separated on anothertwo-dimensional gel and either special silver stained or stained byCoomassie Brilliant Blue. The most acidic of the three spots (theleftmost spot) was well isolated from other proteins and excised fromthe Coomassie Brilliant Blue-stained gel. This spot was digested byendoproteinase Lys-C and analyzed by MALDI-MS. A comparison of theresulting peptide pattern with known human proteins in the SWISS-PROTdatabase revealed a match with human plasma protease C1 inhibitor.

Cleavage of C1 Inhibitor by StcE-His

To confirm the interaction between StcE and human C1 inhibitor and totest the possibility that StcE may proteolyze C1 inhibitor, whole andammonium sulfate-precipitated fractions of human serum were mixed withStcE-His, separated by SDS-PAGE, and transferred to nitrocellulose forimmunoblot analysis. Using an anti-human C1 inhibitor antibody, wedetected the presence of C1 inhibitor in samples lacking StcE-His andthe absence of C1 inhibitor in samples containing StcE-His (FIG. 4). Aspredicted by Jurkat cell aggregation, the 0–30% and 30–60% ammoniumsulfate-precipitated fractions of human serum were enriched for C1inhibitor compared to the 60–100% fraction. After treatment withStcE-His, however, little to no C1 inhibitor could be detected in any ofthe fractions. The addition of EDTA or BPS to the mixture prevented thedisappearance of C1 inhibitor from the serum samples, indicating aspecific requirement for divalent cations, most likely zinc, for StcEactivity (data not shown).

To confirm that the proteolysis of C1 inhibitor was a direct result ofan interaction with StcE-His, we mixed purified human C1 inhibitor withStcE-His and removed aliquots of the reaction at various time points foranalysis by immunoblot. Using an anti-human C1 inhibitor antibody, wedetected the disappearance of a 105 kDa band corresponding tofull-length C1 inhibitor and the appearance of an approximately 60 kDacleavage product in a time-dependent manner (FIG. 5).

Examination of Patient Fecal Filtrates for StcE

Freshly passed stool samples from children with culture-positive E. coliO157:H7 (n=6), Campylobacter jejuni (n=2), Shigella B (n=2), orClostridium difficile (n=2) infections were diluted 1:10 in PBS andpassed through a 0.45 μm filter. Thirty μl of thawed filtrate wassuspended in 1× sample buffer, heated (95–100° C. for 5 min) andelectrophoresed on 8% polyacrylamide gels. Separated proteins weretransferred to nitrocellulose and probed with a polyclonal antibody toStcE-His as described above. Twenty μl of the same samples was added to1×10⁶/ml Jurkat cells in 10% FCS and gentamicin (50 μg/ml) for 24 hoursat 37° C. in 5% CO₂ to determine the ability of the filtrates toaggregate Jurkat cells.

Construction and Analyses of StcE E435D-His Mutant

The StcE E435D-His mutant was created using the PCR-based method ofoverlap extension (Horton et al. 1993). The first two PCR reactions were(i) stcE top strand primer 587 (5′-CCGCTCCGGTGAACTGGAGAATA-3′) (SEQ IDNO:8) with its partner mutagenic primer 592(5′-GACCATAATTATGACCAACATCATGACTGA-3′) (SEQ ID NO:9) and (ii) stcEbottom strand primer 573 (5′-CCTTATCTGCGGAGGCTGTAGGG-3′) (SEQ ID NO:10)with its partner mutagenic primer 574(5′-TGAGTTCAGTCATGATGTTGGTCATAATTAT-3′) (SEQ ID NO:11). Each reactionused 50 pM of each primer, about 100 ng of template DNA, and Deep Ventpolymerase (New England Biolabs) in a 100 μl reaction. The reactionswere run in a thermocycler under appropriate conditions (11) and theresulting products were purified on a 1% agarose gel using the QIA-quickGel Extraction Kit (Qiagen). The next PCR reaction contained 5 μl eachof the gel-purifed fragments, along with the stcE primers 587 and 573and Deep Vent polymerase in a 100 μl reaction. The PCR products weregel-purified as above and cut with the restriction endonucleases PmeIand BsrG1. pWL107 was also cut with PmeI and BsrG1 and the mutant PCRproduct was ligated into pWL107, creating pTEG1. The base substitutionwas confirmed by sequence analysis. pTEG1 was transformed into E. colistrain BL21 (DE3) to create WAM2726 and StcE E435D-His (SEQ ID NO:19)was overexpressed and purified from this strain as described above. Thepurified protein was then analyzed for its ability to aggregate Jurkatcells as described above.

Purified C1-INH (one μg) was mixed with or without StcE-His (one μg) orStcE E435-His (one μg) overnight at room temperature in 500 μl bufferAD, precipitated with TCA (to 10%), electrophoresed on an 8%polyacrylamide gel, and transferred to nitrocellulose before analysis byimmunoblot with an anti-C1-INH antibody as described above.

Purified C1-INH (500 ng) and human serum (50 μg) were electrophoresed onan 8% polyacrylamide gel in duplicate and the separated proteins weretransferred to nitrocellulose for Far Western analysis. Essentially thesame protocol was followed as described above with the followingdifference: one blot was probed with purified StcE-His (2 μg/ml) and theother with purified StcE E435D-His (2 μg/ml).

Colony Blot Analysis

A one kb fragment of stcE was PCR amplified from pO157 using the primersstcE5′846 (5′-GAGAATAATCGAATCACTTATGCTC-3′) (SEQ ID NO:12) andstcE3′1773 (5′-CGGTGGAGGAACGGCTATCGA-3′) (SEQ ID NO:13) under standardreaction conditions. The PCR product was purified on a 1% agarose gelusing the QIA-quick Gel Extraction Kit (Qiagen) and fluorescein-labeledusing the ECL random prime labeling system (Amersham Life Science).Bacterial strains from the DEC collection, EDL933, and EDL933cu werepatched onto sterile Magna Lift nylon transfer membranes (Osmonics) onLB plates and grown overnight at room temperature. Colonies were lysedby placing the membranes on 3 MM Whatman paper soaked in 0.5 M NaOH.Neutralization was performed by placing the membranes first on 3 MMWhatman paper soaked in 1 M Tris, pH 7.5 and then on 3 MM Whatman papersoaked in 0.5 M Tris, pH 7.5/1.25 M NaCl. DNA was then crosslinked usinga UV stratalinker. The blots were pre-hybridized in Church buffer (0.5 Mdibasic sodium phosphate, pH 7.3, 7% SDS, 1% BSA, 1 mM EDTA) at 65° C.for one hour before the addition of the labeled probe. Hybridizationproceeded overnight at 65° C. The membranes were then washed at 65° C.in 1×SSC/0.1% SDS for 15 minutes and then in 0.5×SSC/0.1% SDS for 15minutes. The membranes were incubated with an anti-fluorescein labeled,HRP-conjugated antibody. The membrane was developed using the LumiGLOChemiluminescent Substrate Kit (Kirkegaard and Perry Laboratories).

PCR Analysis of StcE

Oligonucleotides were designed to amplify by PCR regions of stcE tocover the length of the ˜2.8 kbp promoter and gene. Primers stcE5′1(5′-TTTACGAAACA-GGTGTAAATATGTTATAAA-3′) (SEQ ID NO:14) and stcE3′845(5′-CAGTTCACCG-GAGCGGAACCA-3′) (SEQ ID NO:15) covered the first third,stcE5′846 and stcE3′1773 covered the middle third, and stcE5′1774(5′-GCTTCAGC-AAGTGGAATGCAGATAC-3′) (SEQ ID NO:16) and stcE3′2798(5′TTATTTAT-ATACAACCCTCATTGACCTAGG-3′) (SEQ ID NO:17) covered the finalthird. Genomic DNA was isolated from E. coli strains DEC3A-E, DEC4A-E,DEC5A-E, EDL933, and EDL933cu using the Wizard Genomic DNA PurificationKit (Promega) as per the manufacturer's instructions. PCR was performedusing 20 pM of each primer, about 100 ng of template DNA, and Deep Ventpolymerase (New England Biolabs) in a 100 μl reaction. The reactionswere run in a thermocycler under standard conditions. PCR products wereelectrophoresed on 1% agarose gels and visualized with ethidium bromide.

Isolation and Analyses of Bacterial Culture Supernatants

E. coli strains DEC3A-E, DEC4A-E, DEC5A-E, EDL933, and EDL933cu weregrown in Lennox L broth at 37° C. overnight. Culture supernatants wereharvested by centrifugation at 4° C. for 15 minutes at 10,000×g andfiltered through a 0.45 μm filter. Supernatants were precipitated withammonium sulfate to 75% saturation. The precipitates were centrifugedfor 15 minutes at 16,000×g at 4° C. and resuspended in buffer AD. Theresuspended precipitates were dialyzed against three changes of ADbuffer overnight to remove residual ammonium sulfate.

Purified C1-INH (one μg) was mixed with 200 μl of ammoniumsulfate-precipitated culture supernatants at room temperature overnightin a total volume of 500 μl buffer AD before precipitation with TCA (to10%) and electrophoresis on 8% polyacrylamide gels. Separated proteinswere transferred to nitrocellulose and immunoblot analysis was performedwith an anti-C1-INH antibody as described above. Culture supernatantsalone were separated on 8% polyacrylamide gels and transferred tonitrocellulose before immunoblot analysis was performed using ananti-StcE-His antibody as described above.

Specificity of StcE-His for C1-INH

To evaluate the specificity of StcE-His, potential target proteins(listed in Table 2), target protein (2 μg) was mixed with eitherStcE-His (1.28 μg) or Pseudomonas aeruginosa elastase (20 ng)(Calbiochem EC# 3.4.24.26) overnight at 37° C. in 500 μl buffer AD (20mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 0.01% Tween-20) andprecipitated with TCA (to 10%) prior to electrophoresis on 8–10%polyacrylamide gels. Proteins in the gels were then Coomassie-stained ortransferred to nitrocellulose for immunoblot analysis as above.

StcE is able to cleave both purified and serum-associated C1-INH. OnlyC1-INH was cleaved by StcE-His; the sizes and staining intensities ofall other potential substrates were the same in the presence and absenceof StcE-His. In contrast, elastase degraded most of the proteins tested.Elastase treatment of C1-INH normally produces an inactive 95 kDaproduct (12), whereas treatment of C1-INH with StcE-His results in˜60–65 kDa C1-INH fragment(s) (FIG. 6). This indicates that the StcEcleavage site of C1-MNH is distinct from that used by elastase. Weemployed a sensitive fluorimetric assay based on the digestion of aBODIPY FL-labeled casein substrate (EnzChek, Molecular Probes) toanalyze further the ability of StcE-His to act as a non-specificendoprotease. Serial two-fold dilutions of StcE-His or P. aeruginosaelastase were mixed with the casein substrate per the manufacturer'sinstructions before fluorescent measurement of casein degradation.StcE-His was unable to degrade casein even at high proteinconcentrations (up to 6.4 μg/unit of volume), while elastase was able toact on casein at lower concentrations (range: 0.5 ng to 1 μg/unit ofvolume) (data not shown).

TABLE 2 Proteolysis of substrates incubated with StcE-His or P.aeruginosa elastase Substrate StcE Elastase C1 inhibitor (CortexBiochem, San Leandro, CA) + + α2-antiplasmin (Calbiochem, San Diego, CA)− + α1-antitrypsin (Sigma, St. Louis, MO) − + α1-antichymotrypsin(Sigma, St. Louis, MO) − + antithrombin (Enzyme Research Labs, SouthBend, IN) − + α2-macroglobulin (Calbiochem, San Diego, CA) − + vonWillebrand factor (gift from Dr. D. Mosher, − N.D. UW-Madison) collagenIV (Rockland, Gilbertsville, PA) − − fibronectin (Calbiochem, San Diego,CA) − + serum albumin (New England Biolabs, Beverly, MA) − N.D. IgA1(Cortex Biochem, San Leandro, CA) − + Elastin (Sigma, St. Louis, MO) − +Gelatin (BioRad, Hercules, CA) − + N.D. = not done Two μg of theindicated protein substrates were mixed with 1.28 μg StcE-His or 20 ngP. aeruginosa elastase overnight at 37° C. prior to electrophoresis bySDS-PAGE and staining with Coomassie Brilliant Blue. StcE was unable todigest any of the proteins tested other than C1-INH, while P. aeruginosaelastase had activity against a broad range of targets.

Detection of StcE in Feces

The Shiga-like toxin has been identified in the feces of patientsinfected with E. coli O157:H7 (13, 14). To demonstrate that StcE isproduced in vivo during an E. coli O157:H7 infection, we examined fecalfiltrates collected from patients with E. coli O157:H7 and non-E. coliO157:H7-mediated diarrhea for the presence of StcE antigen and activity.Twelve fecal samples were diluted in PBS and filtered before analysis byimmunoblot with polyclonal antibodies to StcE-His. A strongly reactiveband with a molecular weight similar to StcE was present in the filtratefrom one child infected with E. coli O157:H7 (FIG. 7, sample 2). BecauseStcE is able to mediate the aggregation of T cells, we examined theability of the twelve fecal filtrates to aggregate Jurkat cells. Twentyμl of each filtrate was added to 5×10⁵ Jurkat cells in the presence of10% FCS. The one sample that contained a StcE-reactive speciesaggregated Jurkat cells to the same extent as 50 ng/ml purifiedStcE-His; all other samples were negative in this assay, even after 24hours of incubation (data not shown).

StcE Contains a Zinc Metalloprotease Active Site.

As the predicted StcE amino acid sequence has a consensus Zn²⁺-ligandbinding site of metalloproteases (434: HEVGHNYGLGH) (SEQ ID NO:3), weexamined the possibility that the glutamic acid residue at position 435is critical for the proteolysis of C1-INH. This amino acid in other zincmetalloproteases acts as the catalytic residue for proteolysis (15)(16), and other researchers have shown that a conservative amino acidsubstitution from glutamic to aspartic acid disrupts the activity of theprotease while maintaining its structure (15). By introducing a singlechange in the sequence of stcE at base 1442 from an A to a T, we createdthe same mutation and examined the ability of the recombinant mutant(StcE E435D-His) to digest C1-INH. While StcE-His is able to degradeC1-INH, we observed no such cleavage with the mutant protein (FIG. 8A)under the same conditions. The results of Far Western analysis (FIG. 8B)suggested that StcE E435D-His is unable to bind to C1-INH (or asimilarly sized protein in human serum), which seemed to suggest thatglutamic acid 435 is necessary for both binding and cleavage of C1-INH.The StcE-mediated aggregation of Jurkat cells was also affected by theE435D mutation. Jurkat cells will aggregate in response to StcE-His atconcentrations as low as 1 ng/ml, while cells treated with as much as200 ng/ml StcE E435D-His did not aggregate (data not shown). Thus, theglutamic acid residue at position 435 is critical for StcE-mediatedaggregation of Jurkat cells, as well as proteolysis of C1-INH. However,based on subsequent results of studies involving StcE potentiation ofC1-INH-mediated protection of cells, described in detail below, itappears that StcE E435D-His can in fact interact with C1-INH.

Detection of StcE Among Diarrheagenic E. coli Strains.

In order to establish the prevalence of stcE among other pathogenicstrains of E. coli, we examined the Diarrheagenic E. coli (DEC)collection, a reference set of 78 E. coli strains provided by Dr. TomWhittam of the University of Pennsylvania, for the presence of stcE.This collection contains a variety of enterohemorrhagic,enteropathogenic, and enterotoxigenic E. coli strains of differentserotypes isolated from humans, non-human primates, and other mammalsthat are associated with disease symptoms, including diarrhea,hemorrhagic colitis, or HUS. The DEC collection is divided into 15subgroups based on electrophoretic type, which is indicative of thegenetic similarity of one strain to another. By using colony blotanalysis, we found that all O157:H7 strains of E. coli (DEC3 and DEC4)contain DNA that hybridizes with an internal one kb region of stcE(Table 3). Surprisingly, three of five enteropathogenic O55:H7 strainsof E. coli (DEC5A, C, & E) also hybridized with the stcE probe. BecauseO157:H7 strains are thought to have evolved from an 055:H7 predecessor,this result suggests a source of the stcE gene for current O157:H7strains of E. coli. None of the other strains in the DEC collectionhybridized with the stcE probe by colony blot analysis.

To confirm the presence of the gene among the stcE-positive groups inthe DEC collection, we isolated genomic DNA from DEC3A-E, DEC4A-E,DEC5A-E, EDL933, and EDL933cu and used oligonucleotide pairs designed toamplify regions of stcE by PCR. Three primer sets were chosen to amplifystcE and its promoter from bases 1–845, 846–1773, and 1774–2798. Anappropriately-sized PCR product was amplified with all three primerpairs from EDL933, DEC3A-E, and DEC4A-E (FIG. 9). Appropriately sizedproducts were obtained with primer pairs 846–1773 and 1774–2798 for DEC5A, C, and E, but there were no products with primer pair 1–845 fromthese strains. It is possible that this region of stcE, which includesthe putative promoter, is sufficiently different from stcE found onpO157 to prevent priming and amplification. DEC5B and D were negativefor all three reactions.

TABLE 3 Incidence of stcE and its product in the DEC collection DECPredominant Disease Number of Number of Number Serotype Category stcEpositive StcE positive 1A–E O55:H6 EPEC 0/5 ND 2A–E O55:H6 EPEC 0/5 ND3A O157:H7  EHEC + + 3B O157:H7  EHEC + + 3C O157:H7  EHEC + + 3DO157:H7  EHEC + + 3E O157:H7  EHEC + + 4A O157:H7  EHEC + + 4B O157:H7 EHEC + + 4C O157:H7  EHEC + + 4D O157:H7  EHEC + + 4E O157:H7  EHEC + +5A O55:H7 EPEC + + 5B O55:H7 EPEC − − 5C O55:H7 EPEC + − 5D O55:H7 EPEC− − 5E O55:H7 EPEC + + 6A–E O111:H12 EPEC 0/5 ND 7A–E O157:H43 ETEC 0/50/5 8A–E O111:H8  EHEC 0/5 ND 9A–E  O26:H11 EHEC 0/5 ND 10A–E  O26:H11EHEC 0/5 ND 11A–E O128:H2  EPEC 0/5 ND 12A–E O111:H2  EPEC 0/5 ND 13A–EO128:H7  ETEC 0/5 ND 14A–E O128:H21 EPEC 0/5 ND 15A–E O111:H21 EPEC 0/5ND Using a one kb probe internal to stcE, colony blot analyses wereperformed to determine which strains in the DEC collection containedstcE. Strains that were positive for the gene were checked for secretionof StcE as well proteolytic activity against C1-INH. Strains in boldcontained the gene and produced the protein. Strains in italicscontained the gene but lacked detectable protein. All other strains inthe DEC collection were negative for stcE. ND = not done.

Because previous experiments showed that StcE is released into theculture medium by EDL933 (FIG. 3), we examined whether the stcE-positivestrains from the DEC collection also release StcE into the culturemedium. We grew DEC3A-E, DEC4A-E, DEC5A-E, EDL933, and EDL933cuovernight in Lennox L broth at 37° C., harvested and concentrated theculture supernatants 100-fold. By immunoblot analysis we were able todetect StcE-reactive antigen in the supernatants of all stcE-positivestrains and DEC5C (FIG. 10). The intensity of the reactive band variedfrom strain to strain and seemed to be stronger in the DEC3 group. Totest if the bacterial-conditioned culture supernatants contained C1-INHproteolytic activity, we mixed purified C1-INH with the supernatantsovernight and examined substrate cleavage by immunoblot. Again, allstcE-positive strains except DEC5C were able to degrade C1-INH (FIG.11). Interestingly, DEC5B converted C1-INH from a single band to adoublet; this is unlikely to be related to StcE activity and thesignificance of this is unknown. It appears that DEC5C is unable torelease StcE into the culture medium, although it contain stcE-like DNA.This may be due to a lack of expression of the gene or release of theprotein from the cell.

II. Potentiation of C1-INH by StcE

Bacterial Strains, Buffers, and Materials.

All chemicals were purchased from Sigma (St. Louis, Mo.) unlessotherwise indicated. Buffers used were PBS (10 mM sodium phosphate, 140mM NaCl, pH 7.4), TBS (20 mM Tris, 150 mM NaCl, pH 7.5), VBS (5 mMveronal, 145 mM NaCl, pH 7.4), VBS²⁺ (VBS containing 0.15 mM CaCl₂ and0.5 mM MgCl₂), and VBS containing 10 mM EDTA. Recombinant DNAmanipulations were performed by standard methods. pTB4 was constructedby amplifying bases 207–2795 of the stcE gene from pO157 by PCR with theprimers stcESac5′207 (5′-CCGAGCTCCGGCTGATAATAATTCAGCCATTTATTTC-3′) (SEQID NO:20) and stcE3′Xba2795 (5′-CCTCGAGTTTATATACAACCCTGATTG-3′) (SEQ IDNO:21) and cloning the product into the SacI-XhoI sites of pET24d(+)(Novagen, Madison, Wis.). WAM2751 is E. coli strain BL21(DE3) (Novagen)transformed with pTB4. pTB5 was constructed in the same manner as pTB4by amplifying the equivalent bases of the stcE E435D mutant from pTEG1,as described above. WAM2804 is E. coli strain BL21(DE3) transformed withpTB5. StcE′-His, lacking the StcE N-terminal signal sequence andcontaining a 6×His tag at the C-terminus, and StcE′ E435D-His, a similarprotein with a single amino acid change at residue 435 (glutamic toaspartic acid) that disrupts the catalytic activity of the protease,were purified from WAM2751 and WAM2804, respectively, as described aboveand dialyzed into VBS, pH 6.5. Purified C1-INH was obtained fromAdvanced Research Technologies (San Diego, Calif.) and both purifiedkallikrein and C1s from Calbiochem (San Diego, Calif.). All proteinswere stored at −80° C. Monoclonal antibodies (mAbs) 3C7 and 4C3 weregenerous gifts from Dr. Philip A. Patston (University of Illinois atChicago, Ill.).

Hemolytic Assays.

Sheep erythrocytes were prepared according to Mayer (42). Erythrocyteswere opsonized with an anti-sheep red blood cell Ab for 10 min prior touse. Human serum (0.5%) was mixed with opsonized erythrocytes (1×10⁷ in50 μl) in VBS²⁺ to a total volume of 200 μl for one hour at 37° C.before the addition of one ml VBS+10 mM EDTA to stop complementactivity. To measure the amount of hemoglobin released by lysed cells,erythrocytes were pelleted and the O.D.₄₁₂ of the supernatant wasmeasured in a spectrophotometer. The percent lysis was determined bysubtracting the O.D.₄₁₂ in the absence of serum and dividing by themaximum possible O.D.₄₁₂ obtained by lysis of erythrocytes in water.Where indicated, increasing concentrations of StcE′-His or bovine serumalbumin (BSA) were incubated with serum overnight at room temperaturebefore the start of the assay. To determine the effect of StcE-treatedC1-INH on erythrocyte lysis, increasing concentrations of StcE′-His orBSA were mixed with C1-INH (16 μg), or increasing concentrations ofC1-INH were mixed with StcE′-His (one pg) or StcE′ E435D-His (one μg) ina total volume of 149 μl VBS²⁺ overnight at room temperature before thestart of the assay. Statistical analyses were performed by the unpairedt test.

Flow Cytometry.

C1-INH (8μg) was untreated or treated with StcE′-His (one μg) in a totalvolume of 149 μl VBS²⁺ as described above before the addition ofopsonized sheep erythrocytes and human serum deficient in complementcomponent C5 (Quidel, San Diego, Calif.). Erythrocytes were incubatedfor 10 minutes at 37° C. before the addition of VBS+10 mM EDTA. Cellswere washed with VBS²⁺ and incubated on ice for 30 min with polyclonalgoat-anti-human IgG against C1-INH (Cedarlane Laboratories).Erythrocytes were washed, incubated on ice for 30 min withFITC-conjugated rabbit-anti-goat IgG, and resuspended in VBS²⁺ foranalysis by flow cytometry using a fluorescence-activated cell sorter(FACSCalibur, Becton Dickinson, San Jose, Calif.). Where indicated,StcE′-His was removed from the assay mixture by adsorption to Ni-NTAagarose beads (Qiagen, Valencia, Calif.) in the presence of 50 mMimidazole prior to the addition of sheep erythrocytes. To measureStcE-treated C1-INH saturation kinetics of erythrocytes, increasingconcentrations of C1-INH were mixed with StcE′ E435D-His (one μg) beforethe addition of sheep erythrocytes (1×10⁷) as described above in theabsence of human serum and analyzed by flow cytometry.

To determine if StcE binds sheep erythrocytes, StcE′-His was labeled viaits primary amines with the Alexa Fluor 488 dye as described by themanufacturer (Molecular Probes, Eugene, Oreg.). Sheep erythrocytes(5×10⁶) were opsonized as described above, pelleted, and resuspended in500 μl VBS²⁺ before the addition of StcE′-His or Alexa-labeled StcE′-His(250 ng) for 10 minutes at 37° C. Erythrocytes were pelleted and washedwith VBS²⁺ before analysis by flow cytometry. To measure the point atwhich erythrocytes become saturated with StcE, increasing concentrationsof Alexa-labeled StcE′-His were added to sheep erythrocytes (1×10⁷) for10 minutes at 37° C. before analysis by flow cytometry as describedabove.

Immunoblot Analyses.

Purified, activated C1s (1.5 μg) was untreated, treated with C1-INH (100ng), or treated with C1-INH in the presence of StcE′-His or StcE′E435D-His (50 ng each) for one hour at 37° C. in a total volume of 30 μlVBS²⁺. An equal volume of non-reducing sample buffer was then added, thesamples were heated to 95–100° C. for 5 minutes, and the proteinsseparated on an 8% SDS-PAGE gel. Proteins were transferred tonitrocellulose and analyzed by immunoblot as described using apolyclonal goat anti-C1s Ab (Calbiochem).

In other experiments, mAb 4C3 was coupled to Protein A-sepharose beadsas described (43) and used to remove trace amounts of reactive centerloop (RCL)-cleaved C1-INH from the purified C1-INH preparation. VirginC1-INH (one μg) was then incubated with or without StcE′-His (one μg) orkallikrein (2 μg) for 18 hours at room temperature beforeelectrophoresis on an 8% reducing SDS-PAGE gel. Separated proteins weretransferred to nitrocellulose and analyzed by immunoblot as describedusing a polyclonal anti-human C1-INH Ab (Serotec, Raleigh, N.C.), mAb3C7, or mAb 4C3.

Cell Culture.

COS-7 cells (a gift from Dr. Donna Paulnock, University of Wisconsin,Madison, Wis.) were cultured in DMEM (Invitrogen, Carlsbad, Calif.) with10% heat-inactivated fetal calf serum (FCS) (Mediatech, Herndon, Va.),non-essential amino acids, and penicillin/streptomycin/amphotericin B(Invitrogen). Cells were transfected with either hC1-INH/pcDNA3.1(−) orC-serp(98)/pcDNA3.1(−) (generous gifts from Dr. Alvin E. Davis, HarvardUniversity, Cambridge, Mass.) using cationic lipids (Lipofectamine PLUS,Invitrogen). After transfection, cells were cultured in the presence ofG418 (Invitrogen). Recombinant proteins were metabolically labeled with[³⁵S]-methionine (Amersham Biosciences, Piscataway, N.J.) for 24 hoursbefore immunoprecipitation.

Immunoprecipitation.

Culture supernatants (100 μl) from C1-INH-transfected COS-7 cells weretreated with StcE′-His (10 μg) overnight at room temperature beforeincubation with polyclonal goat anti-human C1-INH IgG (CedarlaneLaboratories, Ontario, Canada) and Protein A-sepharose (2 hours, roomtemperature). Pellets were washed three times with TBS, resuspended insample buffer, and electrophoresed on 10% reducing SDS-PAGE gels. Gelswere fixed, dried, and visualized with a phosphorimager (Typhoon 8600,Amersham Biosciences). In other experiments, C1-INH (5 μg) was untreatedor treated with StcE′-His or StcE′ E435D-His (5 μg each) for 10 minutesat 37° C. in 500 μl of buffer (100 mM Tris, pH 8.0) before the additionof polyclonal goat anti-human C1-INH IgG. The mixture was rotated for 30minutes at 4° C., after which 20 μl of a Protein A-sepharose slurry wasadded for 2 hours. The Protein A-sepharose beads were subsequentlywashed three times in buffer before immunoblot analysis with ananti-StcE Ab.

Kallikrein Activity Assay.

Increasing concentrations of C1-INH were mixed with StcE′-His (250ng) ina total volume of 100 μl assay buffer (50 mM Tris, pH 8.0, 100 mM NaCl)at room temperature overnight after which EDTA was added to 5 mM to stopthe reaction. Purified kallikrein was diluted to 100 ng in 50 μl assaybuffer and mixed with C1-INH for one hour at 37° C. before adding thechromogenic substrate S-2302 (Chromogenix, Milan, Italy) to each tube.Tubes were incubated at room temperature for 30 minutes beforedetermining the absorbance of the substrate at 410 nm in aspectrophotometer. Percent kallikrein activity was determined bysubtracting the O.D.₄₁₀ in the absence of kallikrein and dividing by themaximum possible O.D.₄₁₀ obtained by kallikrein activity in the absenceof C1-INH.

Serum Resistance.

C1-INH (8 μg) was untreated or treated with StcE′-His (one μg) in atotal volume of 176 μl VBS²⁺ overnight at room temperature, after whichhuman serum was added to 2%. E. coli K-12 strain C600 was grown to anO.D.₅₉₅ of 0.5 in LB broth at 37° C. with aeration before being washedonce and resuspended with an equivalent volume of VBS²⁺. Bacteria (20μl) were added to the reactions, incubated at 37° C. for one hour, and10 μl aliquots were mixed with VBS+10 mM EDTA to stop complementactivity. Ten-fold serial dilutions of bacteria were plated on LB agarand percent survival was determined by dividing CFUs by the total numberof bacteria after one hour in the absence of serum. Statistical analysiswas performed by the unpaired t test.

Inhibition of Classical Complement-Mediated Erythrocyte Lysis by StcE.

Previous research from our laboratory demonstrated that StcE, ametalloprotease secreted by E. coli O157:H7, cleaves the serum proteinC1-INH from its apparent M_(r) of 105 kDa to produce ˜60–65 kDa species.Because C1-INH is an essential regulator of the classical complementpathway, we examined the effect of StcE on the classicalcomplement-mediated lysis of sheep erythrocytes. Human serum was mixedovernight with increasing concentrations of StcE or the control proteinBSA before adding to opsonized sheep erythrocytes for one hour at 37° C.The reaction was stopped with EDTA and the amount of hemoglobin releasedby lysed erythrocytes into the supernatant was measured.

Serum alone lysed 75.0% (±2.7% SEM) of erythrocytes, and BSA had noeffect on the ability of serum to lyse erythrocytes (FIG. 12). At higherconcentrations, StcE significantly reduced classical complement-mediatederythrocyte lysis compared to equivalent amounts of BSA (2 μg, p<0.01; 4μg, p<0.005).

StcE Potentiates C1-INH-Mediated Inhibition of Classical Complement.

Although StcE cleaves the serum component C1-INH, the data representedin FIG. 12 does not implicate the serpin directly in StcE-mediatedclassical complement inhibition. To examine if StcE-treated C1-INH playsa role in this process, we mixed purified C1-INH with increasingconcentrations of StcE or BSA overnight and added this mixture to humanserum and opsonized sheep erythrocytes for one hour at 37° C. As before,the reaction was stopped with EDTA and the amount of hemoglobin releasedby lysed erythrocytes into the supernatant was measured. The addition of0.4 IU untreated C1-INH to the assay decreased erythrocyte lysis from82.9% (±1.1% SEM) to 33.7% (±1.8% SEM) (FIG. 13 A), demonstrating theeffective role of C1-INH in the inhibition of classical complementactivity. Whereas BSA-treated C1-INH was unchanged in inhibitoryactivity, 0.4 IU StcE-treated C1-INH reduced erythrocyte lysis tobetween 1.5% (±0.5% SEM) and 0.1% (±0.1% SEM) of total.

To confirm the effect of StcE on C1-INH-mediated inhibition oferythrocyte lysis, increasing concentrations of C1-INH were untreated ortreated with StcE′-His overnight prior to the addition of human serumand opsonized sheep erythrocytes. Increasing concentrations of untreatedC1-INH (0.05 to 0.4 IU) resulted in a dose-dependent decrease inerythrocyte lysis, ranging from 76.5% (±2.4% SEM) to 27.5% (±1.1% SEM)(FIG. 13B). However, in the presence of one μg StcE′-His, the sameconcentrations of C1-INH significantly reduced lysis below that ofuntreated C1-INH (ranging from 46.7% (±1.5% SEM) lysis to 2.8% (±1.3%SEM) lysis, all p values<0.0005). These results demonstrate the directrole of StcE-treated C1-INH in the decrease of classicalcomplement-mediated erythrocyte lysis. Additional experiments confirmthat StcE-treated C1-INH continues to react with its natural targets C1rand/or C1s to mediate the inhibition of classical complement,maintaining the target specificity of the serpin (data not shown).

StcE Binds Erythrocyte Surfaces.

In order to understand how StcE might potentiate C1-INH, we asked ifStcE could interact with erythrocytes, thereby acting as a bindingprotein for C1-INH on cell surfaces. Unlabeled StcE′-His or a form ofStcE′-His fluorescently labeled via its primary amines with the AlexaFluor 488 dye (Alexa-StcE′-His) (250 ng each) were added to opsonizedsheep erythrocytes for 10 minutes at 37° C. Erythrocytes were pelletedand washed with VBS²⁺ before analysis by flow cytometry. Erythrocytestreated with Alexa-labeled StcE′-His showed 80-fold greater meanfluorescence compared to cells treated with unlabeled StcE′-His (FIG. 14A), demonstrating a direct interaction between these cells and theprotease. Furthermore, we observed that StcE continues to bind sheeperythrocytes even at lower temperatures (0–4° C.), suggesting that thisinteraction is not mediated by an active cellular process (data notshown). To determine if the interaction between erythrocytes and StcE isspecific and therefore saturable, we mixed increasing concentrations ofAlexa-labeled StcE′-His with sheep erythrocytes (1×10⁷) as describedabove. We observed that this number of erythrocytes becomes saturatedwith StcE′-His at 3.2 μg of the protease in 500 μl (FIG. 14B). Based onthe calculated molecular weight of StcE′-His, at this concentration weestimate approximately 1.8×10⁶ molecules of StcE are bound pererythrocyte.

StcE Localizes C1-INH to Erythrocyte Surfaces.

Based on the ability of StcE to directly bind erythrocytes, we asked ifStcE could localize C1-INH to erythrocyte surfaces, thereby possiblyincreasing the local concentration of inhibitor at the site of potentiallytic complex formation. C1-INH was untreated or treated with StcE′-Hisovernight before the addition of opsonized sheep erythrocytes andC5-deficient human serum (to prevent formation of the membrane attackcomplex and lysis of the cells) for 10 min at 37° C. Erythrocytes werewashed, treated with an Ab against C1-INH and washed again prior to theaddition of a FITC-conjugated secondary Ab. Deposition of C1-INH wassubsequently analyzed by flow cytometry. Little to no C1-INH binding wasmeasured on erythrocytes treated with only StcE′-His or native C1-INHcompared to mock-treated cells (FIG. 15A). Increased deposition ofC1-INH was detected on erythrocytes mixed with 0.2 IU StcE-treatedC1-INH, however. Similar results were observed in the absence of humanserum, indicating that serum components or complement activation are notrequired for the localization of StcE-treated C1-INH to erythrocytesurfaces. As we observed with the interaction between StcE and sheeperythrocytes, StcE-treated C1-INH continues to bind the cells at 0–4° C.(data not shown).

The results suggest that StcE may directly mediate the binding of C1-INHto the cell surface. To test this possibility, C1-INH was incubated withor without StcE′-His overnight as described earlier, following whichNi-NTA agarose beads were added to the samples in the presence of 50 mMimidazole to specifically remove the 6×His-tagged StcE protein from theassay. The agarose beads were pelleted and the supernatants were mixedwith sheep erythrocytes and analyzed by flow cytometry as describedabove. In the absence of the protease, StcE-treated C1-INH no longerbinds to erythrocyte surfaces (FIG. 15B), indicating that StcE itself isrequired to sequester C1-INH to erythrocytes. The absence of StcE′-Hisfrom the Ni-NTA agarose-treated samples and the presence of equivalentamounts of C1-INH between Ni-NTA agarose-treated and untreated sampleswere confirmed by immunoblot analyses (data not shown).

To determine the level at which erythrocytes become saturated withStcE-treated C1-INH, we mixed StcE′ E435D-His (a mutant form of theprotein containing a single amino acid change from glutamic to asparticacid at position 435 that is unable to cleave C1-INH with increasingconcentrations of C1-INH (from 0.25–16 μg) before the addition ofopsonized sheep erythrocytes as described above. We chose to use StcE′E435D-His in this experiment so as to measure the saturation of sheeperythrocytes with C1-INH without the creation of StcE-cleaved C1-INH,which might reduce the levels of the serpin bound to the cell surface,thereby increasing the amount needed to saturate the cells. We observedthat, in the presence of one μg StcE′ E435D-His, this number oferythrocytes becomes saturated with C1-INH at 4 μg, or 0.1 IU, of theserpin (FIG. 15C). Based on the observed molecular weight of matureC1-INH and assuming uniform binding of the primary and secondaryantibodies to their antigens, at this concentration we estimateapproximately 2.25×10⁶ molecules of C1-INH are bound per erythrocyte.Finally, to determine if C1-INH and StcE can interact in solution (priorto binding erythrocytes), C1-INH was mixed with either StcE′-His orStcE′ E435D-His for 10 min at 37° C. before immunoprecipitating themixture with an anti-C1-INH Ab. After separating the immunoprecipitatedproteins by SDS-PAGE and transferring them to nitrocellulose, bothStcE′-His and StcE′ E435D-His were detected with an anti-StcE′-His Ab,demonstrating that a complex of StcE and C1-INH can be formed insolution (FIG. 15D).

Cleavage of C1-INH by StcE is not Necessary to Protect Cells AgainstComplement Activity.

To test if the proteolysis of C1-INH by StcE is necessary to provideerythrocytes increased protection against classical complement activityover that of untreated C1-INH, we mixed C1-INH with either StcE′-His orthe proteolytically inactive StcE′ E435D-His. After overnightincubation, the samples were added to human serum and opsonized sheeperythrocytes for one hour at 37° C. before determining the amount ofhemoglobin released into the supernatant by the lysed cells as describedabove. In the presence of 0.2 IU C1-INH, human serum lysed 75.8% (±1.5%SEM) of the erythrocytes, whereas StcE′-His-treated C1-INH significantlydecreased erythrocyte lysis to 25.4% (±3.5% SEM, p<0.005) (FIG. 16A).Interestingly, the cleavage of C1-INH by StcE is not required for theprotection of erythrocytes from complement activity, as StcE′E435D-His-treated C1-INH was able to significantly reduce the lysis ofthe cells to 16.7% (±1.3% SEM, p<0.005). The difference in the extent oferythrocyte lysis between the StcE′-His-treated and the StcE′E435D-His-treated C1-INH samples was not significant (p>0.05).

To determine if the subsequent cleavage of C1-INH by StcE affects thebinding of the serpin to erythrocytes, we incubated C1-INH (2 μg) withStcE′-His or StcE′ E435D-His (one μg each) overnight before assessingthe levels of surface-associated C1-INH by flow cytometry as describedabove. Indeed, the levels of C1-INH in the presence of StcE′ E435D-Hison erythrocyte surfaces are 22-fold higher than in the presence ofStcE′-His (FIG. 16B), suggesting that, cleaved C1-INH binds erythrocytesless efficiently than intact C1-INH. In total, the data presented inFIGS. 4 and 5 demonstrate that the increased protection of erythrocytesby StcE-treated C1-INH is dependent upon the physical presence of StcEand not the cleavage of C1-INH by the protease.

StcE is Unable to Potentiate C1-INH in the Absence of Cells.

Data presented so far demonstrate the ability of StcE to localize C1-INHto erythrocytes, thereby providing increased complement-inhibitingactivity at the cell surface. To determine if this potentiation canoccur in solution (i.e. in the absence of cells), we measured whetherStcE affects the ability of C1-INH to inhibit kallikrein, anotherC1-INH-regulated molecule, by monitoring the cleavage of a chromogenicsubstrate of kallikrein, S-2302(H-D-Prolyl-L-phenylalanyl-L-arginine-p-nitroaniline dihydrochloride).Increasing concentrations of C1-INH were untreated or treated withStcE′-His overnight at room temperature, after which the samples wereallowed to react with kallikrein for one hour at 37° C. TheC1-INH/kallikrein mixtures were subsequently incubated with S-2302 for30 min at room temperature before determining total kallikrein activityby measuring the change in absorbance of the samples in aspectrophotometer. For the purpose of this assay, kallikrein in theabsence of C1-INH was considered to be 100% active. As expected,increasing concentrations of C1-INH resulted in a dose-dependentdecrease in kallikrein activity, ranging from 88.8% (±2.6% SEM) to 10.3%(±1.3% SEM) activity (FIG. 17A). The addition of StcE-treated C1-INH tothe assay did not significantly alter the inactivation of kallikreincompared to untreated C1-INH.

We also examined the ability of StcE-treated C1-INH to interact with anexcess of C1s in solution, thereby forming an SDS-insoluble complex.Purified, activated C1s (1.5 μg) was mixed with C1-INH (100 ng) andStcE′-His or StcE′ E435D-His (50 ng each) for one hour at 37° C. beforeseparating the proteins by non-reducing SDS-PAGE and analyzing themixture by immunoblot with an anti-C1s Ab. The high molecular weightband in samples containing C1-INH that are absent from the samplecontaining C1s alone are indicative of the C1s-C1-INH interaction. IfStcE were able to potentiate C1-INH in solution, an increase in theintensity of the C1s-C1-INH band would be visible; however, this doesnot appear to be the case (FIG. 17B). Thus, the mechanism ofStcE-mediated potentiation of C1-INH is dependent on the presence ofcell surfaces upon which the protease-serpin complex can bind.

Interaction of StcE with the N-terminus of C1-INH.

The ability of StcE to interact with C1-INH while maintaining theinhibitory activity of the molecule suggests that StcE may bind C1-IN inthe heavily glycosylated N-terminal domain, leaving the serpin domainunaffected. Therefore, to further characterize the site(s) of cleavageby StcE, we examined StcE-treated C1-INH with the monoclonal antibodies3C7 and 4C3, directed against the amino terminus of C1-INH (44) and theRCL-inserted form of C1-INH (45), respectively. As most preparations ofpurified C1-INH contain trace amounts of RCL-cleaved C1-INH, we removedthis species of C1-INH from the mixture by immunoprecipitation with mAb4C3 prior to analysis. Virgin C1-INH was treated with StcE orkallikrein, a serine protease inactivated by C1-INH via its interactionwith and cleavage of the RCL, prior to analysis by immunoblot with apolyclonal anti-human C1-INH Ab (FIG. 18A, left panel), 3C7 (FIG. 18A,middle panel), or 4C3 (FIG. 18A, right panel). As expected, analysiswith 3C7 detected both virgin C1-INH and kallikrein-reacted C1-INH, butdid not detect StcE-cleaved C1-INH, indicating a modification of theC1-INH N-terminus by StcE. Additionally, analysis with 4C3 detectedRCL-inserted C1-INH produced upon interaction with kallikrein, but notvirgin C1-INH or StcE-treated C1-INH.

To confirm that StcE interacts with the N-terminal domain of C1-INH, weexamined the ability of StcE to cleave a recombinant C1-INH proteinlacking this region. Coutinho et al. demonstrated that C-serp(98), arecombinant C1-INH molecule lacking the N-terminal amino acids 1–97 andcontaining only the serpin domain, binds its serine protease substratesidentically to wild-type C1-INH and is effective in inhibiting C1activity in hemolytic assays (31). We expressed recombinant, full-lengthhuman C1-INH (hC1-INH) and C-serp(98) in COS-7 cells and harvested theculture media after 24 hours in the presence of [³⁵S]-methionine.Samples were untreated or treated with StcE′-His overnight beforeimmunoprecipitating metabolically labeled protein with polyclonalanti-human C1-INH IgG. Both hC1-INH and C-serp(98) migrated at theappropriate molecular weights on a reducing SDS-PAGE gel, however, onlyhC1-INH was cleaved by StcE′-His; C-serp(98) was unaffected by theprotease (FIG. 18B). These analyses further support the evidence thatStcE does not inactivate C1-INH, but instead interacts with the heavilyglycosylated N-terminal domain of C1-INH, leaving the serpin domainavailable for interaction with C1-INH targets.

Increased Bacterial Serum Resistance in the Presence of StcE-TreatedC1-INH.

StcE is secreted by E. coli O157:H7, a human pathogen that may come incontact with blood or blood products during the course of an infection.Based on its ability to enhance C1-INH-mediated inhibition of classicalcomplement, we examined if StcE could provide serum resistance to E.coli. As E. coli O157:H7 is naturally serum resistant and contains avariety of factors that could contribute to its protection fromcomplement (1), we chose to assess the role of StcE-treated C1-INH inthe survival of a serum-sensitive strain of E. coli. E. coli K-12 strainC600 was grown to mid-log phase, pelleted, and resuspended in anequivalent amount of VBS²⁺ before the addition of human serum and 0.2 IUC1-INH or StcE-treated C1-INH. Bacteria were incubated at 37° C. for onehour, serially diluted and plated onto LB agar to determine the numbersof surviving CFUs. In the presence of human serum alone, 0.07% (±0.06%SEM) of bacteria survived, demonstrating the exquisite serum sensitivityof E. coli strain C600 (FIG. 19). The addition of StcE′-His to bacteriaat the beginning of the assay had no significant effect on survival(0.04% survival, ±0.03% SEM). As expected, the addition of untreatedC1-INH increased survival of bacteria to 3.9% (±0.9% SEM). The additionof StcE-treated C1-INH to the assay, however, caused a significantincrease in bacterial survival over untreated C1-INH (16.5% survival,±1.9% SEM; p <0.005), indicating a contribution to complement resistanceby StcE.

III. Further Characterization of StcE and its Role in Pedestal FormationConstruction of StcE Knockout and Restored Strains

Construction of EDL933 ΔstcE::cm (WAM 2815).

Construction of EDL933 ΔstcE::cm (WAM 2815). A deletion mutant of stcEfrom EDL933 was constructed by the linear recombination (λRed) method ofDatsenko and Wanner (77). Briefly, the oligonucleotides 5′ 707 (5′-ATGAAATTAAAGTATCTGTCATGTACGATCCTTGCCCCTTGTGTAGGCTGGAGC TGCTTC-3′) (SEQ IDNO:22) and 3′ 708 (5′-TAATTTATATACAACCCTCATTGACCTAGGTTTACTGAAGCATATGAATATCCTCCTTAG -3′) (SEQ ID NO:23) were used toamplify by PCR the chloramphenicol resistance cassette from thenon-polar (in frame, with added ribosome binding site) plasmid templatepKD3. The resulting product was then transformed by electroporation intoWAM2806 (EDL933 carrying pKD46, grown at 30° C. in the presence of 10 mMarabinose). Transformants cured of pKD⁴⁶ and lacking the stcE codingsequence were selected by growth on LB agar containing chlormaphenicol(20 μg/ml) at 42° C. and were confirmed by PCR. More than 95% of thecoding sequence of stcE was deleted, leaving behind the 5′ and 3′ endsof the gene encoded by the oligonucleotides.

A StcE complemented strain having the stcE gene restored was createdusing a Tn7 transposase system similar to that described by DeLoney etal. (77). The stcE gene was PCR amplified using primers 5′1135 (5′-AAGGGC CCC TCT GAG GTG TCT GTT AAA CCC GTG G-3′) (SEQ ID NO:24) and 5′1136(5′-AAA AA TGG CCA CGA AGT GGC CGC ACC GTC TCA GG-3′) (SEQ ID NO:25).The gene was put into the ApaI-MscI sites of pEVS107 and electroporatedinto the mating strain WAM1301, E. coli 517λpir, creating a straincalled WAM2980. The strains WAM2980, WAM2815, and WAM2871 (E. colicarrying the helper plasmid pUX-BF13 which has the Tn7 transposase genestnsABCDE), were mated. This resulted in strain WAM2997, which is theWAM2815 ΔstcE::cm strain that carries a single copy of stcE on thechromosome and has restored StcE expression.

Production of an Untagged, Purified StcE Protein

Recombinant, untagged StcE protein was created using the IMPACT proteinexpression system from NEB (New England Biolabs, Beverly, Mass.).Briefly, the stcE gene was amplified by PCR using Deep Vent polymerase(NEB) and purified pO157 plasmid DNA as a template. The gene was theninserted into pTYB1 (NEB) at the NheI and SapI restriction sites of themultiple cloning site, creating pTEG11. This plasmid created a fusiongene of stcE with sequences encoding a chitin binding domain and anintein protease. The plasmid was moved into the expression strain ER2566(NEB). The chitin-binding domain of the expressed protein allowedaffinity purification on a chitin-sepharose column, while the inteinprotease allows the target protein to be released from the two fusiondomains. The result was a StcE protein that had three extra residues(Met-Ala-Ser) at its N-terminus, but is otherwise identical to StcEsecreted from strains carrying the pO157 plasmid. This same protocol canbe used to express a proteolytically inactive form of rStcE calledE435D, which has a mutation of a glutamate to aspartate residue at thezinc metalloprotease.

Tissue Culture of HEp-2 Cells.

HEp-2 cells, which are derived from a contaminant of the human cervicalepithelial cell line HeLa, were maintained in Eagle's modified medium(Mediatech Herndon, Va.) supplemented with 10% FBS (Atlanta Biologicals,Norcross, Ga.), 10 mM sodium pyruvate, penicillin, streptomycin, andamphotericin B. Cells were passed after achieving confluence by liftingwith 0.25% Trypsin-EDTA (Mediatech) and diluting to 1:5 or 1:10

Microscopic Analysis of Pedestal Formation

Evaluation of pedestal formation was adapted from Knutton et al.Infection & Immunity 57: 1290 (1989) and Methods in Enzymology 253: 324.

Bacterial strains were inoculated from a single colony on an LB plateinto Lennox broth (2 ml) at 37° C., without shaking and grown tostationary phase (12–18 hours). The media used for infection wasDubelco's modified MEM medium supplemented as above. The overnightbacterial culture was diluted 1:25 in this media, for an averageinoculation of 8×10⁶ CFU/ml. HEp-2 cells from a confluent dish werediluted 1:5 or 1:10 and grown in 4 or 8 well slides (Nalge NuncInternational, Naperville, Ill.) for 24 to 48 hours (50–80% confluent).Cells were washed three times in PBS to remove any residual antibioticsin the media. Then the DMEM-bacterial mixture was placed on the cells,0.5 ml for 4 well slide, 0.25 ml for 8 well slide, and incubated at 37°C. for 6–7 hours, with or without simultaneous addition of recombinantStcE (2 μg). Media was removed at midpoint of incubation and replacedwith fresh media and no additional bacteria.

After infection, cells were washed thoroughly (5–6 times) with PBS andfixed in paraformaldhehyde (3% for 10 minutes). Cells were washed withPBS (three times for two to three minutes) after fixation. A 15 minuteblocking step with antibody dilution solution (2% BSA, 0.1% Triton X-100in PBS) was used to inhibit non-specific staining. Bacteria were stainedwith a monoclonal antibody recognizing the O157 antigen (U.S.Biologicals, Swampscott, Mass.) for 30 minutes in a 1:200 dilution.After washing in PBS, cells were stained with the secondary antibodysolution containing goat anti-rabbit conjugated to Alexa488, 1:1000(Molecular Probes, Netherlands), and Phalloidin conjugated to Alexa5941:400 (Molecular Probes).

Samples were analyzed using a Zeiss (Carl Zeiss MicroImaging Inc.,Thornwood, N.Y.) fluorescent microscope using a 40× plan apochromat NA1.3 objective. Images were acquired with a Axiocam monochrome CCD cameraand Openlab software (Improvision, Lexington, Mass.). Random fields fromblinded sample wells were selected out-of-focus on the phalloidinchannel such that no actin pedestals were discernible that might biasselection. Number of foci that had formed pedestals were counted in eachfield after 10 (8 well slides) or 20 (4 well slides) fields were imagedof each sample. Foci were defined as either a single bacterium or acluster of bacteria separated from other foci by more than the length ofa bacterium.

The results indicate that the StcE knockout mutant has a lower capacityfor forming pedestals than the wild type or the complemented strain, butthe ability is restored by supplementation with exogenous StcE (FIG.20).

StcE Reduces Viscosity of Saliva and Solubilizes Mucus

Because StcE has the capacity to cleave a heavily glycosylated,mucin-like region of C1-INH, studies were undertaken to determinewhether StcE was capable of cleaving other substrates. Human saliva andsputum, which are good sources of mucins and glycoproteins, wereevaluated as follows.

Whole human saliva was collected and split into 10 ml samples. Theviscosity of the samples was measured before and after a 3 hourtreatment with rStcE or buffer. Both the treatment and measurements tookplace at 37° C. Relative viscosity was measured in a Cannon-FenskeRoutine Viscometer (range: 0.5 to 4 cP) (Cannon Instrument Company,State College, Pa.), and elution time was measured, with less viscoussolutions that travel through the viscometer relatively quickly havingshorter elution times than more viscous solutions. Treatment with StcEwas found to reduce the viscosity of saliva by about 66% (FIG. 21).

Mucus from a person with a cold was expectorated into a sample tube.Aliquots (1 ml) were transferred into a 10 ml glass tube using a bluetip (made for a p-1000 pipette) with the tip cut off, creating a widerbore. One tube was untreated and to the other StcE (40 μg) was added.Both were vortexed briefly (setting 4 of 9) and incubated 3 days at roomtemperature. The samples were then vortexed briefly and tested forability to be pipetted by a blue tip with a 1000 μl pipet and forability to flow freely in the 10 ml sample tube. The StcE treated samplewas free-flowing and easily pipetted while the untreated sample wasstill thick and viscous, and would get stuck in a blue tip.

Identification of Saliva Proteins Cleaved by StcE

Whole human saliva was collected and treated with rStcE (1 μg) oruntreated for one hour at room temperature. Proteins were separated bySDS-PAGE (8% acrylamide) and stained with Coomassie R-250. Withreference to FIG. 22, lane 1 includes rStcE-His; lane 2 includesuntreated saliva; and lane 3 contains saliva treated with rStcE-His.Proteins of interest (indicated with errors) were excised from the gel,digested with trypsin and analyzed by MADLI-TOF mass spectrometry.Peptide masses were compared to the ProteinProspector database(University of California at San Francisco) for identification. Theproteins were found to be MUC7 and gp-340/DBMT1.

ELISA for C1-INH Cleavage by StcE

An enzyme-linked immunosorbant assay (ELISA) was used to test for StcEprotease activity toward C1-INH in a 96 well format. The primaryantibody 3C7 (gift of Phil Patston, University of Illinois at Chicago)binds only uncut C1-INH, but does not bind to either fragment producedby StcE cleavage. The antibody is thought to bind somewhere in theN-terminal 100 amino acids of C1-INH, but the binding site is presumedto be destroyed by StcE cleavage.

First, 0.1 to 10 mmol C1-INH was mixed with 0–5 mmol StcE in a totalvolume of 100 μl PBS (phosphate buffered saline) in a round bottommicrotitre plate (Sarstedt). After 1 hour at 37° C., an EDTA solution(10 mM in PBS, 50 μl) was added to stop the reaction. The reactionmixtures were transferred to an ELISA plate (Dynatech flatbottom Immulonplates, Alexandria, Va.) and held at room temperature (RT) for 2 hoursto allow binding of proteins to the plate. The wells were emptied,bovine serum albumin (BSA, 1% in PBS, 100 μl) was added to each well andincubated for 30 minutes to block non-specific binding. The wells werethen washed three times for one minute each with PBST (PBS with 0.05%Tween-20, 200 μl). The primary antibody was diluted 1:1500 in blockingsolution and 100 μl was added to each well and incubated at RT for 60minutes. Wells were again washed, and then the secondary antibody (goatanti-mouse conjugated to horseradish peroxidase, BioRad, Hercules,Calif.) was diluted 1:3000 in blocking solution and 100 μl added to eachwell and incubated at RT for 30 minutes. After a final wash, substratesolution (100 μl TMB, BioRad) was added to each well. After a ten-minuteincubation while rocking at RT, stop solution was added (100 μl 1Nsulfuric acid). Absorbance at 450 nm was read and used to calculate theC1-INH remaining in the wells. C1-INF remaining was plotted as afunction of the amount of C1-INH added to the well (FIG. 24). Theresults indicate that the amount of detectable (i.e., intact) C1-INH isreduced in the presence of StcE.

This method provides a means of detecting the presence of StcE activityin a test sample. In addition, the method provides a means forevaluating the ability of a test substance to inhibit cleavage of C1-INHin the presence of StcE.

Proposed ELISA for Detection of StcE Protein in Mixtures or ClinicalSamples

StcE is expressed in enterohemorrhagic E. coli infections, although itis presently known whether the protein is expressed early or late in thecourse of colonization and infection. If it expressed early, detectionof StcE could be a valuable tool for early detection of infection.

A clinical sample such as a fecal filtrate or bacterial supernatant isadsorbed onto an untreated ELISA plate. After a block and wash step, thewells are probed with an antibody against the StcE protein. Followinganother wash, a secondary antibody (e.g., anti-mouse conjugated tohorseradish peroxidase, BioRad) is added to the wells. After a finalwash, a substrate solution, such as TMB substrate (BioRad), is cleavedby the horseradish peroxidase, and its product is detected, indicatingthe presence of StcE.

Alternatively, a sandwich ELISA method is employed in which the ELISAplate is be pre-adsorbed with antibodies to StcE. The subsequent stepsare the same as those outlined in the preceding method.

Model of StcE Role in EHEC Colonization of Host Cells

We propose a model in which the StcE-mediated increase in pedestalformation is due to cleavage of proteins from the glycocalyx and/or cellsurface, allowing a closer interaction of bacterium and host cell.Cleavage of these proteins may also lead to decreased adherence ofnormal flora, which would compete with EHEC for space and resources.Addition of StcE to cell extracts prepared from uninfected HEp-2 cellsshowed that StcE changed the banding pattern of proteins separated viaSDS-PAGE. This data supports the model that host cell proteins are beingcleaved or modified in a way that is favorable to intimate adherence byEHEC. It is expected that most of these proteins are cell-surfaceproteins, and identification of these potential substrates is underway.

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1. A method of reducing the viscosity of a material comprising a mucinor a glycosylated polypeptide comprising contacting the material with aviscosity reducing effective amount of a polypeptide having at least 95%amino acid identity to amino acid residues 24–886 of SEO ID NO:2 andretaining the ability to cleave mucin or a glycosylated polypeptide. 2.The method of claim 1, wherein the material is saliva.
 3. The method ofclaim 1, wherein the material is sputum.
 4. The method of claim 1,wherein the polypeptide comprises amino acid residues 24–886 of SEQ IDNO:2.